Microbial delivery system

ABSTRACT

The present invention provides methods and compositions for treating or preventing allergic responses, particularly anaphylactic allergic responses, in subjects who are allergic to allergens or susceptible to allergies. Methods of the present invention utilize administration of microorganisms to subjects, where the microorganisms produce allergens and protect the subjects from exposure to the allergens until phagocytosed by antigen-presenting cells. Particularly preferred microorganisms are gram-negative bacteria, gram-positive bacteria, and yeast. Particularly preferred allergens are proteins found in foods, venoms, drugs and latex that elicit allergic reactions and anaphylactic allergic reactions in individuals who are allergic to the proteins or are susceptible to allergies to the proteins. The proteins may also be modified to reduce the ability of the proteins to bind and crosslink IgE antibodies and thereby reduce the risk of eliciting anaphylaxis without affecting T-cell mediated Th1-type immunity.

PRIORITY INFORMATION

The present application is a divisional of U.S. patent application Ser.No. 09/731,375 entitled “Bacterial Polypeptide Delivery” filed Dec. 6,2000 which claims priority under 35 U.S.C. 119(e) to U.S. ProvisionalPatent Application Ser. No. 60/195,035 entitled “Bacterial PolypeptideDelivery” filed Apr. 6, 2000.

RELATED APPLICATIONS

The present invention is generally in the area of controlled delivery ofantigens for use in vaccination or induction of tolerance to allergens,and in particular relates to cellular delivery of proteins andpolypeptides. This application is related to U.S. Ser. No. 60/169,330entitled “Controlled Delivery of Antigens” filed Dec. 6, 1999; U.S. Ser.No. 09/141,220 entitled “Methods and Reagents for Decreasing ClinicalReaction to Allergy” filed Aug. 27, 1998; U.S. Ser. No. 09/455,294entitled “Peptide Antigens” filed Dec. 6, 1999; U.S. Ser. No. 09/494,096filed Jan. 28, 2000 entitled “Methods and Reagents for DecreasingClinical Reaction to Allergy” by Bannon et al.; and U.S. Ser. No.09/527,083 entitled “Immunostimulatory Nucleic Acids and Antigens” byCaplan filed Mar. 16, 2000; the teachings of which are all incorporatedherein by reference in their entirety.

BACKGROUND OF THE INVENTION

Allergic reactions pose serious public health problems worldwide. Pollenallergy alone (allergic rhinitis or hay fever) affects about 10-15% ofthe population, and generates huge economic costs. For example, reportsestimate that pollen allergy generated $1.8 billion of direct andindirect expenses in the United States in 1990 (Fact Sheet, NationalInstitute of Allergy and Infectious Diseases; McMenamin, Annals ofAllergy 73:35, 1994). Asthma, which can be triggered by exposure toantigens, is also a serious public health problem, and like anaphylacticallergic reactions, can lead to death in extreme cases. Asthma currentlyaccounts for millions of visits yearly to hospitals and is increasing infrequency. The only treatment currently available is for alleviation ofsymptoms, for example, to relieve constriction of airways. More seriousthan the economic costs associated with pollen and other inhaledallergens (e.g., molds, dust mites, animal danders) is the risk of ananaphylactic allergic reaction observed with allergens such as foodallergens, insect venoms, drugs, and latex.

Allergic reactions result when an individual's immune system overreacts,or reacts inappropriately, to an encountered antigen. Typically, thereis no allergic reaction the first time an individual is exposed to aparticular antigen. However, it is the initial response to an antigenthat primes the system for subsequent allergic reactions. In particular,the antigen is taken up by antigen presenting cells (APC; e.g.,macrophages and dendritic cells) that degrade the antigen and thendisplay antigen fragments to T cells. T cells, in particular CD4⁺“helper” T-cells, respond by secreting a collection of cytokines thathave effects on other immune system cells. The profile of cytokinessecreted by responding CD4⁺ T cells determines whether subsequentexposures to the antigen will induce allergic reactions. Two classes ofCD4⁺ T cells (Th1 and Th2) influence the type of immune response that ismounted against an antigen.

Th2 cells can secrete a variety of cytokines and interleukins includingIL-4, IL-5, IL-6, IL-10 and IL-13. One effect of IL-4 is to stimulatethe maturation of B cells that produce IgE antibodies specific for theantigen. Allergic responses to allergens are characterized by theproduction of antigen-specific IgE antibodies which are dependent onhelp from IL-4 secreting CD4⁺ T cells. These antigen-specific IgEantibodies attach to receptors on the surface of mast cells, basophilsand eosinophils, where they act as a trigger to initiate a rapidallergic reaction upon the next exposure to antigen. When the individualencounters the antigen a second time, the antigen is quickly bound bythese surface-associated IgE molecules. Each antigen typically has morethan one IgE binding site, so that the surface-bound IgE moleculesquickly become crosslinked to one another through their simultaneous(direct or indirect) associations with antigen. Such cross-linkinginduces mast cell degranulation, resulting in the release of histaminesand other substances that trigger allergic reactions. Individuals withhigh levels of IgE antibodies are known to be particularly prone toallergies.

Current treatments for allergies involve attempts to “vaccinate” asensitive individual against a particular allergen by periodicallyinjecting or treating the individual with a crude suspension of the rawallergen. The goal, through controlled administration of known amountsof antigen, is to modulate the IgE response mounted in the individual.If the therapy is successful, the individual's IgE response isdiminished, or can even disappear. However, the therapy requires severalrounds of vaccination, over an extended time period (3-5 years), andvery often does not produce the desired results. Moreover, certainindividuals suffer anaphylactic reactions to the vaccines, despite theirintentional, controlled administration.

Clearly, there is a need for treatments and preventive methods forpatients with allergies to allergens that elicit serious allergicresponses including anaphylaxis.

SUMMARY OF THE INVENTION

The present invention provides methods and compositions for modulatingthe immune response in a subject. It is an aspect of the presentinvention to provide a method of treating or preventing undesirableallergic reactions and anaphylactic allergic reactions to allergens in asubject. Methods of the present invention involve administering tosubjects, microorganisms that express or produce allergens of interest.Without being limited to the proposed mechanism of action, afteradministration the microorganisms are taken up by antigen-presentingcells in the subject where the expressed antigens are released. Afterbeing processed inside the antigen-presenting cells and displayed on thecell surface, the processed antigens activate T-cell mediated immuneresponses. Use of genetically modified microorganisms to express anddeliver allergens to a subject therefore reduces the exposure of theallergens to the subject's IgE antibodies, which lead to allergicreactions and possibly anaphylaxis. The present invention thereforereduces the risk of anaphylaxis during immunotherapy. Furthermore, themicroorganisms may act as a natural adjuvant to enhance desirableTh1-type immune responses.

In a preferred embodiment, microorganisms are genetically modified toexpress selected polypeptides or proteins, and are used as deliveryvehicles in accordance with the present invention. Such microorganismsinclude but are not limited to bacteria, viruses, fungi (includingyeast), algae, and protozoa. Generally, preferred microorganisms for usein accordance with the present invention are single cell, single sporeor single virion organisms. Additionally, included within the scope ofthe present invention are cells from multi-cellular organisms which havebeen modified to produce a polypeptide of interest.

In a particularly preferred embodiment, bacteria or yeast are used asmicroorganisms to express and deliver allergenic proteins to individualsto treat or prevent allergic responses, including anaphylactic allergicresponses, to the allergens. Gram-positive and gram-negative bacteriamay be used in the present invention has delivery vehicles. Antigensexpressed by the bacteria may be secreted or non-secreted. Secretion ofproteins may involve secretion into the cellular medium. Forgram-negative bacteria and yeast, secretion may involve secretion intothe periplasm. Secretion of polypeptides may be facilitated by secretionsignal peptides. In certain preferred embodiments microorganismsexpressing allergenic compounds may be administered to subjects incompositions as attenuated microorganisms, non-pathogenicmicroorganisms, non-infectious microorganisms, or as killedmicroorganisms. Preferably, the killed microorganisms are killed withoutdegrading the antigenic properties of the polypeptides.

In another preferred embodiment, the allergens utilized are allergensfound in foods, venom, drugs and a rubber-based products. Particularlypreferred protein allergens are found in foods and venoms that elicitanaphylactic allergic responses in subjects who are allergic to theallergens. Included in the present invention are peptides andpolypeptides whose amino acid sequences are found in the proteinsallergens in nature. Also included in the present invention areallergens that have modifications that reduce the ability of thepeptides, polypeptides and proteins to bind and crosslink IgEantibodies. Also included in the present invention are non-peptideallergens that are produced by microorganisms and include for exampleantibiotics such as penicillin.

In another aspect of the invention, compositions for use in treating orprevent allergic and anaphylactic allergic responses in a subjectcomprise microorganisms that have been engineered by the hand of man,and preferably by the introduction of one or more introduced nucleicacids, to produce allergens in accordance with the present invention. Incertain preferred embodiments, the produced allergens are peptides,polypeptides, or proteins encoded by the introduced nucleic acids(s).

BRIEF DESCRIPTION OF FIGURES

FIG. 1. Experiments designed to determine the optimal temperature forheat-killing bacteria (E. coli) are depicted in graphic form. The numberof surviving colonies in aliquots of samples are shown as a function oftemperature (Celsius).

FIG. 2. Determination of protein produce per cell. The optical density(O.D.) of the HIS-tagged Ara h 2 allergen was determined from animmunoblot where different concentrations of E. coli extract has beenelectrophoresed on SDS-PAGE gels. The allergen O.D. was used to estimatethe amount of protein produced by that extract.

FIG. 3. Results of ELISA analysis of Ara h 2-specific IgG antibodiesproduced in mice following injection of E. coli producing Ara h 2. IgG1is on the left and IgG2a is on the right.

FIG. 4. Results of ELISA analysis of Ara h 3-specific IgG antibodiesproduced in mice following injection of E. coli producing Ara h 3. IgG1is on the left and IgG2a is on the right.

FIG. 5 shows an example of how IgE binding epitopes were mapped to aspecific amino acid sequence on the Ara h 1 allergen. In particular,FIG. 5 depicts twenty-two 10-mer peptides (SEQ ID NOs. 45-66) that spanamino acid residues 82-133 (SEQ ID NO. 44) of the Ara h 1 allergen (SEQID NO. 2). This region of the Ara h 1 allergen includes epitopes 4, 5,6, and 7, as identified in Table 1.

FIG. 6 shows an example of how IgE binding epitopes were mapped to aspecific amino acid sequence on the Ara h 2 allergen. In particular,FIG. 6 depicts seven 10-mer peptides (SEQ ID NOs. 68-74) that span aminoacid residues 55-76 (SEQ ID NO. 67) of the Ara h 2 allergen (SEQ ID NO.4). This region of the Ara h 2 allergen includes epitopes 6 and 7 asidentified in Table 2.

FIG. 7 shows an example of how IgE binding epitopes were mapped to aspecific amino acid sequence on the Ara h 3 allergen. In particular,FIG. 7 depicts six 15-mer peptides (SEQ ID NOs. 76-81) that span aminoacid residues 299-321 (SEQ ID NO. 75) of the Ara h 3 allergen (SEQ IDNO. 6). This region of the Ara h 3 allergen includes epitope 4 asidentified in Table 3.

FIG. 8 shows the effect the modified Ara h 2 protein has on IgE binding.

FIG. 9 shows the results of T cell proliferation assays using thewild-type and modified Ara h 2 protein.

DEFINITIONS

“Allergen”: An “allergen” is an antigen that (i) elicits an IgE responsein an individual; and/or (ii) elicits an asthmatic reaction (e.g.,chronic airway inflammation characterized by eosinophilia, airwayhyperresponsiveness, and excess mucus production), whether or not such areaction includes a detectable IgE response). Preferred allergens forthe purpose of the present invention are peptide, polypeptide andprotein allergens. An exemplary list of protein allergens is presentedas an Appendix. This list was adapted fromftp://biobase.dk/pub/who-iuis/allergen.list (updated on Mar. 1, 2000),which provides lists of known allergens. Other preferred allergens arechemical compounds such as small molecules that are produced byproteins. In some embodiments, an allergen is a subset of antigens whichelicits IgE production in addition to other isotypes of antibodies.

“Allergic reaction”: An allergic reaction is a clinical response by anindividual to an antigen. Symptoms of allergic reactions can affectcutaneous (e.g., urticaria, angioedema, pruritus), respiratory (e.g.,wheezing, coughing, laryngeal edema, rhinorrhea, watery/itching eyes)gastrointestinal (e.g., vomiting, abdominal pain, diarrhea), and/orcardiovascular (if a systemic reaction occurs) systems. For the purposesof the present invention, an asthmatic reaction is considered to be aform of allergic reaction. A decreased allergic reaction ischaracterized by a decrease in clinical symptoms following treatment ofsymptoms associated with exposure to an allergen, which can involverespiratory, gastrointestinal, skin, eyes, ears and mucosal surfaces ingeneral.

“Anaphylactic antigen”: An “anaphylactic antigen” according to thepresent invention is an antigen (or allergen) that is recognized topresent a risk of anaphylactic reaction in allergic individuals whenencountered in its natural state, under natural conditions. For example,for the purposes of the present invention, pollens and animal danders orexcretions (e.g., saliva, urine) are not considered to be anaphylacticantigens. On the other hand, food antigens, insect antigens, drugs, andrubber (e.g., latex) antigens latex are generally considered to beanaphylactic antigens. Food antigens are particularly preferredanaphylactic antigens for use in the practice of the present invention.Particularly interesting anaphylactic antigens are those (e.g., nuts,seeds, and fish) to which reactions are commonly so severe as to createa risk of death.

“Anaphylaxis” or “anaphylactic reaction”, as used herein, refers to animmune response characterized by mast cell degranulation secondary toantigen-induced cross-linking of the high-affinity IgE receptor on mastcells and basophils with subsequent mediator release and the productionof pathological responses in target organs, e.g., airway, skin digestivetract and cardiovascular system. As is known in the art, the severity ofan anaphylactic reaction may be monitored, for example, by assayingcutaneous reactions, puffiness around the eyes and mouth, and/ordiarrhea, followed by respiratory reactions such as wheezing and laboredrespiration. The most severe anaphylactic reactions can result in lossof consciousness and/or death.

“Antigen”: An “antigen” is (i) any compound or composition that elicitsan immune response; and/or (ii) any compound that binds to a T cellreceptor (e.g., when presented by an MHC molecule) or to an antibodyproduced by a B-cell. Those of ordinary skill in the art will appreciatethat an antigen may be collection of different chemical compounds (e.g.,a crude extract or preparation) or a single compound (e.g., a protein).Preferred antigens are peptide, polypeptide or protein antigens. In someembodiments, an antigen is a molecule that elicits production ofantibody (a humoral response) or an antigen-specific reaction with Tcells (a cellular response).

“Antigen presenting cells”: “Antigen presenting cells” or APCs” includeknown APCs such as Langerhans cells, veiled cells of afferentlymphatics, dendritic cells and interdigitating cells of lymphoidorgans. The term also includes mononuclear cells such as lymphocytes andmacrophages which take up polypeptides and proteins according to theinvention. In some embodiments, an antigen presenting cell (an APC) is acell which processes and presents peptides to T cells to elicit anantigen-specific response.

“Attenuation”: “Attenuation” of microorganisms as used herein refers tothe manipulation of the microorganisms so that the microorganisms do notinduce significant toxic reactions in individuals or laboratory testanimals. The manipulations include genetic methods and are well known inthe art.

An epitope is a binding site including an amino acid motif of betweenapproximately six and fifteen amino acids which can be bound by eitheran immunoglobulin or recognized by a T cell receptor when presented byan antigen presenting cell in conjunction with the majorhistocompatibility complex (MHC). A linear epitope is one where theamino acids are recognized in the context of a simple linear sequence. Aconformational epitope is one where the amino acids are recognized inthe context of a particular three dimensional structure. Animmunodominant epitope is one which is bound by antibody in a largepercentage of the sensitized population or where the titer of theantibody is high, relative to the percentage or titer of antibodyreaction to other epitopes present in the same protein.

“IgE binding site”: An IgE binding site is a region of an antigen thatis recognized by an anti-antigen IgE molecule. Such a region isnecessary and/or sufficient to result in (i) binding of the antigen toIgE; (ii) cross-linking of anti-antigen IgE; (iii) degranulation of mastcells containing surface-bound anti-antigen IgE; and/or (iv) developmentof allergic symptoms (e.g., histamine release). In general, IgE bindingsites are defined for a particular antigen or antigen fragment byexposing that antigen or fragment to serum from allergic individuals(preferably of the species to whom inventive compositions are to beadministered). It will be recognized that different individuals maygenerate IgE that recognize different epitopes on the same antigen.Thus, it is typically desirable to expose antigen or fragment to arepresentative pool of serum samples. For example, where it is desiredthat sites recognized by human IgE be identified in a given antigen orfragment, serum is preferably pooled from at least 5-10, preferably atleast 15, individuals with demonstrated allergy to the antigen. Those ofordinary skill in the art will be well aware of useful pooling strategyin other contexts.

“Immunologic inducing agents”: The term “immunological inducing agents”is used herein as agents that prompt the expression of Th1 stimulatingcytokines by T-cells and include factors such as, CD40, CD40 ligand,oligonucleotides containing CpG motifs, TNF, and microbial extracts suchas preparations of Staphylococcus aureus, heat killed Listeria, andmodified cholera toxin.

Immunostimulatory sequences are oligodeoxynucleotides of bacterial,viral or invertebrate origin that are taken-up by APCs and activate themto express certain membrane receptors (e.g., B7-1 and B7-2) and secretevarious cytokines (e.g., IL-1, IL-6, IL-12, TNF). Theseoligodeoxynucleotides containing unmethylated CpG motifs cause briskactivation and when injected into animals in conjunction with antigen,appear to skew the immune response to a Th1-type response. See, forexample, Yamamoto, et al., Microbiol. Immunol. 36, 983 (1992); Krieg, etal., Nature 374, 546-548 (1995); Pisetsky, Immunity 5, 303 (1996); andZimmerman, et al., J. Immunol. 160, 3627-3630 (1998).

“Inducible promoter”: The term “inducible promoter”, as used herein,means a promoter site which is activated directly by the presence orabsence of a chemical agent or indirectly by an environmental stimulussuch as temperature changes. A promoter is the region of DNA at whichthe enzyme RNA polymerase binds and initiates the process of genetranscription.

“Mast cell”: As will be apparent from context, the term “mast cell” isoften used herein to refer to one or more of mast cells, basophils, andother cells having IgE receptors, which when activated by crosslinkingbound IgE molecules, releases histamines, vasodilators, and/or othermediators of allergic responses.

“Microorganisms”: “Microorganisms” as used herein are cells, bacteria,fungi, viruses, algae, and protozoa. Preferred microorganisms can begenetically manipulated to produce a desired polypeptide(s).

“Peptide”: According to the present invention, a “peptide” comprises astring of at least three amino acids linked together by peptide bonds.Inventive peptides preferably contain only natural amino acids, althoughnon-natural amino acids (i.e., compounds that do not occur in nature butthat can be incorporated into a polypeptide chain; see, for example,http://www.cco.caltech.edu/˜dadgrp/Unnatstruct.gif, which displaysstructures of non-natural amino acids that have been successfullyincorporated into functional ion channels) and/or amino acid analogs asare known in the art may alternatively be employed. Also, one or more ofthe amino acids in an inventive peptide may be modified, for example, bythe addition of a chemical entity such as a carbohydrate group, aphosphate group, a farnesyl group, an isofarnesyl group, a fatty acidgroup, a linker for conjugation, functionalization, or othermodification, etc.

A peptide or polypeptide is derived from a protein if the amino acidsequence of the peptide or polypeptide is found within the amino acidsequence of the protein. The sequences are preferably identical but mayhave a sequence homology between approximately 80-100%. It is alsorecognized that amino acid residues may be replaced with other aminoacids residues with similar physical properties such as hydrophobicity,hydrophilicity, charge, aromatic structures and polarity.

“Reduced IgE binding”: An inventive composition or antigen is consideredto have “reduced IgE binding” if it demonstrates a lower level ofinteraction with IgE when compared with unmodified antigen in anyavailable assay. For example, a modified antigen is considered to havereduced IgE binding if (i) its affinity for anti-antigen IgE (assayed,for example, using direct binding studies or indirect competitionstudies) is reduced at least about 2-5 fold, preferably at least about10, 20, 50, or 100 fold as compared with intact antigen; (ii) ability ofthe modified antigen to support cross-linking of anti-antigen IgE isreduced at least about 2-fold, preferably at least about 5, 10, 20, 50,or 100 fold as compared with intact antigen; (iii) mast cells containingsurface-bound anti-antigen IgE degranulate less (at least about 2 fold,preferably at least about 3, 5, 10, 20, 50, or 100 fold less) whencontacted with modified as compared with unmodified antigen; and/or (iv)individuals contacted with modified antigen develop fewer (at leastabout 2 fold, preferably at least about 3, 5, 10, 20, 50, or 100 foldfewer) allergic symptoms, or developed symptoms are reduced in intensitywhen exposed to modified antigens as compared with unmodified antigens.

“Secretion signals”: A secretion signal is any amino acid sequence whichwhen conjugated to a peptide, polypeptide or protein facilitates thetransport of the conjugate fusion proteins across cell membranes. Foruses of secretion signals in microorganisms, transport of fusionproteins involves crossing an inner membrane into the periplasm. It ispreferred that secretion signals also facilitate transport of fusionproteins across an outer membrane into an extracellular medium.Secretion of proteins into the extracellular medium is considered“excretion”.

“Sensitized mast cell”: A “sensitized” mast cell is a mast cell that hassurface-bound antigen specific IgE molecules. The term is necessarilyantigen specific. That is, at any given time, a particular mast cellwill be “sensitized” to certain antigens (those that are recognized bythe IgE on its surface) but will not be sensitized to other antigens.

“Small molecules”: As used herein, the term “small molecule” refers to acompound either synthesized in the laboratory or found in nature.Typically, a small molecule is organic and is characterized in that itcontains several carbon-carbon bonds, and has a molecular weight of lessthan 1500 daltons, although this characterization is not intended to belimiting for the purposes of the present invention. Examples of “smallmolecules” that are allergens include without limitation penicillin,alcohols, and aspirin. Non-organic small molecule allergens includesulfites present in wine, for example.

“Susceptible individual”: According to the present invention, a personis susceptible to an allergic reaction if (i) that person has everdisplayed symptoms of allergy after exposure to a given antigen; (ii)members of that person's genetic family have displayed symptoms ofallergy against the allergen, particularly if the allergy is known tohave a genetic component; and/or (iii) antigen-specific IgE are found inthe individual, whether in serum or on mast cells.

“Th1 response” and “Th2 response”: Certain preferred peptides,polypeptides, proteins and compositions of the present invention arecharacterized by their ability to suppress a Th2 response and/or tostimulate a Th1 response preferentially as compared with their abilityto stimulate a Th2 response. Th1 and Th2 responses are well-establishedalternative immune system responses that are characterized by theproduction of different collections of cytokines and/or cofactors. Forexample, Th1 responses are generally associated with production ofcytokines such as IL-1β, IL-2, IL-12, IL-18, IFNα, IFNγ, TNFβ, etc; Th2responses are generally associated with the production of cytokines suchas IL-4, IL-5, IL-10, etc. The extent of T cell subset suppression orstimulation may be determined by any available means including, forexample, intra-cytoplasmic cytokine determination. In preferredembodiments of the invention, Th2 suppression is assayed, for example,by quantitation of IL-4, IL-5, and/or IL-13 in stimulated T cell culturesupernatant or assessment of T cell intra-cytoplasmic (e.g., by proteinstaining or analysis of mRNA) IL-4, IL-5, and/or IL-13; Th1 stimulationis assayed, for example, by quantitation of IFNα, IFNγ, IL-2, IL-12,and/or IL-18 in activated T cell culture supernatant or assessment ofintra-cytoplasmic levels of these cytokines.

DESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS

The present invention provides compositions and methods for modulatingthe immune response in a subject. It is an aspect of the presentinvention that undesirable allergic immune responses to antigens in asubject are treated or prevented by administering modified cells,virions, or spores (“microorganisms”) that express allergens ofinterest. By using genetically modified microorganisms to express anddeliver allergens, exposure of the allergens to the subject'sIgE-mediated allergic immune response is reduced or eliminated. Withoutlimitation to the mechanisms proposed, it is expected that the modifiedmicroorganisms of the present invention are engulfed byantigen-presenting cells (APCs) such as macrophages and dendritic cellswithout exposing allergens to IgE antibodies. Once inside the APCs, theexpressed allergens are released by lysis of the microorganisms orsecretion of the antigen by the microorganisms. The allergens are thenprocessed, for example through partial digestion by the APCs, anddisplayed on the cell surface.

Once the processed antigens are displayed on the cell surface,activation of the cytotoxic T cell response and helper T cell responsepromotes cellular immune response and Th1-mediated B cell response toprotein allergens. In addition, the processed antigens have a reducedability (or no ability) to bind and crosslink IgE antibodies located onthe surface of mast cells and basophils leading to the release ofhistamines and other vasodilators responsible for allergic and sometimesfatal anaphylactic responses.

Host Microorganisms

Any microorganism capable of expressing (e.g., by expression ofpolypeptide or protein allergens, or by expression of polypeptide orprotein enzymes involved in synthesis of small molecule allergens)allergens may be used as delivery vehicles in accordance with thepresent invention. Such microorganisms include but are not limited tobacteria, viruses, fungi (including yeast), algae, and protozoa.Generally, microorganisms are single cell, single spore or single virionorganisms. Additionally, included within the scope of the presentinvention are cells from multi-cellular organisms which have beenmodified to produce a polypeptide of interest. Microorganisms that canbe genetically manipulated to produce a desired polypeptide arepreferred. (Ausubel et al. Current Protocols in Molecular Biology. Wileyand Sons, Inc. 1999, incorporated herein by reference) Geneticmanipulation includes mutation of the host genome, insertion of geneticmaterial into the host genome, deletion of genetic material of the hostgenome, transformation of the host with extrachromosomal geneticmaterial, transformation with linear plasmids, transformation withcircular plasmids, insertion of genetic material into the host (e.g.,injection of mRNA), insertion of transposons, and chemical modificationof genetic material. Methods for constructing nucleic acids (includingan expressible gene), and introducing such nucleic acids into anexpression system to express the encoded protein are well established inthe art (see, for example, Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989, incorporated herein by reference).

Use of microorganisms such as bacteria and yeast for allergen deliveryin accordance with the present invention offers many advantages overdelivery of allergens that are not encapsulated inside microorganismsfor immunotherapy. Generally, microorganisms, such as bacteria, areknown to act as an adjuvant (for a review, see for example, Freytag etal. Curr Top Microbiol Immunol 236:215-36, 1999). Therefore, use ofmicroorganisms to delivery allergens to subjects, and APCs of subjects,provides protection of the allergen from IgE-mediated allergic responsesand also provides an adjuvant effect which elicits a Th1-type immuneresponse from an individual susceptible to allergic responses. Inaddition, use of non-pathogenic, non-infectious, attenuated and/orkilled microorganisms reduces or eliminates toxicity which may beassociated with allergen delivery vehicles.

In a preferred embodiment, bacteria are used as protein deliverymicroorganisms. Generally, bacteria are classified as gram-negative orgram-positive depending on the structure of the cell walls. Thoseskilled in the art are capable of identifying gram-negative andgram-positive bacteria which may be used to express proteins inaccordance with the present invention. Non-limiting examples of generaand species of gram-negative bacteria include Escherichia coli, Vibrocholera, Salmonella, Listeria, Legionella, Shigella, Yersenia,Citrobacter, Enterobacter, Klebsiella, Morganella, Proteus, Providencia,Serratia, Plesiomonas, Aeromonas. Non-limiting examples of genera andspecies of gram-positive bacteria which may be used in the presentinvention include Bacillus subtilis, Sporolactobacillus, Clostridium,Arthrobacter, Micrococcus, Mycobacterium, Peptococcus,Peptostreptococcus, and Lactococcus.

Gram-negative bacterial systems for use as delivery vehicles are knownand may be used in the present invention. For example, E. coli is awell-studied bacteria, and methods of protein expression in E. coli arewell-established. Most strains of E. coli have the advantage of beingnon-pathogenic since E. coli is found naturally in the gut. Therefore,E. coli is preferred as a delivery vehicle in the present invention. Inaddition, Calderwood et al. (U.S. Pat. No. 5,747,028) utilize Vibriocholerae as a delivery vehicle for production of antigens for use as alive vaccine against infectious organisms. Miller and Mekalanos (U.S.Pat. No. 5,731,196) utilize Salmonella as delivery vehicle forproduction of antigens for use as a live vaccine against infectiousorganisms. Hess et al. (Proc. Natl. Acad. Sci. USA 93:1458-1463, 1996)utilize recombinant attenuated Salmonella which secretes antigenicdeterminants of Listeria as a live vaccine to protect againstlisteriosis. Donner et al. (WO 98/50067) utilize attenuated Salmonellatyphimurium as a gram-negative host for secretion of polypeptides forcontrolling fertility and also teach that other attenuated gram-negativestrains including Yersinia may be used to express and secrete suchpolypeptides.

Gram-positive bacteria have also been studied as delivery vehicles forproteins to modulate an immune response in a subject. WO 97/14806describes the use of Lactococcus to deliver polypeptides into a body toenhance the immune response to the polypeptides. However, WO 97/14806does not teach the use of Lactococcus to treat patients with foodallergies and venom allergies which may result in anaphylaxis

In another preferred embodiment, yeast are used as protein deliverymicroorganisms. It is well known that yeast are amenable to geneticmanipulation to express a protein or proteins of choice (Ausubel et al.supra). Furthermore, in general most yeast are non-pathogenic. Withoutlimitation to these species, two well-characterized species of yeast arethe budding yeast Saccharomyces cerevisiae, and the fission yeast,Schizosaccharomyces pombe. Moreover, the administration of yeast thatexpress protein antigens to alter an immune response has been studied.Duke et al. (U.S. Pat. No. 5,830,463; “Duke”) describe the use of yeastto express proteins after administration of the yeast to a mammal.However, Duke does not teach the use of yeast to treat patients withfood allergies and venom allergies which may result in anaphylaxis.

Microorganisms of the present invention may be administered to a subjectas live or dead microorganisms. Preferably if the microorganisms areadministered as live microorganisms, they are non-pathogenic orattenuated pathogenic microorganisms. For applications of the inventionwhere live microorganisms are administered to individuals, preferablythe microorganisms are attenuated and/or are administered in suitableencapsulation materials and/or as pharmaceutical compositions asvaccines to decrease an individuals immune response to the microorganismand/or allergenic compounds. Generally, attenuation involves geneticallymodifying the infectious pathogenic microorganism to reduce or eliminatethe infectious ability of the microorganism. Preferably, themicroorganism is attenuated such that an individual inoculated with themicroorganism does not suffer any cytotoxic effects from the presence ofthe microorganism. Particularly preferred attenuated microorganisms areinfectious intracellular pathogens which are phagocytosed byantigen-presenting cells in individuals who are exposed to themicroorganism. Examples of microorganisms which are intracellularpathogens include Salmonella, Mycobacterium, Leishmania, Legionella,Listeria, and Shigella.

Microorganisms of the present invention may be administered to subjectsafter killing the microorganisms. Any method of killing themicroorganisms may be utilized that does not greatly alter theantigenicity of the expressed polypeptides. Methods of killingmicroorganism include but are not limited to using heat, antibiotics,chemicals such as iodine, bleach, ozone, and alcohols, radioactivity(i.e. irradiation), UV light, electricity, and pressure. Preferredmethods of killing microorganisms are reproducible and kill at least 99%of the microorganisms. Particularly preferred is the use of heat above50 degrees Celsius for a period of time that kills greater than 99% ofthe cells and preferably 100% of the cells.

Inducible Systems

In another preferred embodiment, the inventive expression of allergensby microorganisms is regulated so that synthesis occurs at a controlledtime after the live microorganism is administered to an individual.Preferably the induction of protein synthesis is regulated so thatactivation occurs after the microorganism(s) is taken up byantigen-presenting cells (APCs) and phagocytosed into the endosome. Adesirable result of this regulation is that production of the allergenof interest occurs inside the APCs and therefore reduces or eliminatesthe exposure of the allergen to IgE molecules bound to the surface ofhistamine-releasing mast cells and basophils. This reduces or eliminatesthe risk of anaphylaxis during administration of microorganisms thatproduce anaphylactic antigens.

Any method of controlling protein synthesis in the microorganism may beused in accordance with the present invention. Preferably, the method ofcontrolling protein synthesis utilizes an inducible promoteroperatively-linked to the gene of interest (e.g., a gene which encodes asignal peptide and protein antigen). Many systems for controllingtranscription of a gene using an inducible promoter are known (Ausubelet al. Current Protocols in Molecular Biology. Wiley and Sons. New York.1999). Generally, inducible systems either utilize activation of thegene or derepression of the gene. It is preferred that the presentinvention utilizes activation of a gene to induces transcription.However, inducible systems using derepression of a gene may also be usedin the present invention. Systems using activation are preferred becausethese systems are able to tightly control inactivation (and hence basallevel synthesis) since derepression may result in low levels oftranscription if the derepression is not tight.

Methods of inducing transcription include but are not limited toinduction by the presence or absence of a chemical agent, inductionusing a nutrient starvation inducible promoter, induction using aphosphate starvation inducible promoter and induction using atemperature sensitive inducible promoter. A particularly preferredsystem for regulating gene expression utilizes tetracycline controllableexpression system. Systems which utilize the tetracycline controllableexpression system are commercially available (see for example, Clontech,Palo Alto, Calif.).

Another particularly preferred system for regulating gene expressionutilizes an ecdysone-inducible expression system which is alsocommercially available (Invitrogen, Carlsbad Calif.). Theecdysone-inducible expression system is based on the ability of ecdysonewhich is an insect hormone, to activate gene expression by binding tothe ecdysone receptor. The expression system utilizes a modifiedheterologous protein containing the ecdysone receptor, a viraltransactivation domain (from VP16) and the retinoid X receptor derivedfrom mammalian cells to bind to a modified ecdysone response element inthe presence of a ligand such as ecdysone or an analog (e.g. muristeroneA, ponasterone A).

It is preferred that inducible systems for use in the present inventionutilize inducing agents that are non-toxic to mammalians cells includinghumans. Furthermore, it is preferred that transcriptional inducingagents permeate cells membranes. More specifically for activation ofprotein synthesis in microorganisms after phagocytosis by APCs,transcriptional inducing agents must be able to pass through cellsmembranes of the APC and cell membranes of the microorganism to activatethe expression of genes encoding protein allergens in accordance withthe present invention. Since both tetracycline and ecdysone are able topass through cell membranes and are non-toxic, tetracycline-induciblesystems and ecdysone-inducible systems are ideally suited for use in thepresent invention. However, the use of inducible systems in the presentinvention is not limited to those systems.

It is also preferred that bacteria that have not been phagocytosed arekilled before induction of genes expressing polypeptide allergens ofinterest. A preferred method of killing bacteria is to use antibioticswhich are not permeable to mammalian cell membranes such that onlybacteria that are not phagocytosed are killed. The use of antibiotics inaccordance with the present embodiment reduces or eliminates theproduction of polypeptides by bacteria outside antigen presenting cells.It is important to reduce or eliminate exposure of allergen-producingbacteria to the immune system, especially bacteria that secretepolypeptides, which could elicit a potentially lethal anaphylacticreaction in an individual. Those having ordinary skill in the art arereadily aware of antibiotics which may be used. Such antibiotics includebut are not limited to penicillin, ampicillin, cephalosporin,griseofulvin, bacitracin, polymyxin b, amphotericin b, erythromycin,neomycin, streptomycin, tetracycline, vancomycin, gentamicin, andrifamycin

Secretion Signals

In another embodiment of the present invention, expressed allergens(and/or immunomodulatory molecules, such as cytokines; see below) aresecreted by the microorganisms. Preferably, secretion of the allergensoccurs inside a mammalian cell to reduce or eliminate exposure ofallergens to a subject's allergic immune response. Secretion ofpolypeptides includes secretion into the extracellular medium andsecretion of polypeptides into the periplasm of microorganisms such asgram-negative bacteria and yeast. Advantages of secreting allergens intothe periplasm include reducing leakage of the allergens prior tophagocytosis of the microorganism. This advantage is most applicable innon-inducible systems. Advantages of secreting allergens into theextracellular medium in inducible systems include maximizing the amountof allergens available for processing by antigen-presenting cells afterphagocytosis of the microorganisms of the present invention.

To express secreted polypeptides in bacteria, a variety of bacterialsecretion signals known in the art may be used. For example, theSec-dependent process in E. coli is one which is well known (for areview see Driessen et al. Curr. Opin. Microbiology 1:216-22). Inaddition, the OmpA signal peptide in E. coli has been described by Wongand Sutherland (U.S. Pat. No. 5,223,407). Fusion proteins containingeither of these secretion signal peptides are not fully secreted by thebacteria, but rather transported across the inner membrane of thegram-negative bacteria into the periplasm. These secretion signals maybe used in the present invention to transport allergenic orimmunomodulatory polypeptides into the periplasm of bacteria. Afteradministration of the genetically engineered bacteria to an individualand subsequent phagocytosis by APCs, the allergenic or immunomodulatorypolypeptides in the periplasm are released after degradation of theouter membrane by enzymes in the endosome of the APCs. Preferably, thebacteria synthesize and secrete the polypeptides into the periplasm andare killed, preferably heat-killed, before administration. However, itis recognized that attenuated bacteria may be used to secrete inventiveallergens into the periplasm and administered to individuals.

In another preferred embodiment of secreted proteins or polypeptides,fusion proteins containing secretion signal sequences and allergenic orimmunomodulatory sequences are fully secreted into the extracellularmedium by a microorganism after synthesis of the protein. Such secretionsignals include those found in hemolysin and listeriolysin. In aparticularly preferred embodiment, the hemolysin complex of E. coli isused to transport allergenic or immunomodulatory polypeptides across theinner and outer membrane of a microorganism (e.g. E. coli, Salmonella,Shigella, Vibrio, Yersinia, Citrobacter, Serratia, Pseudomonas) into theextracellular medium (Spreng et al. Mol. Microbiol. 31:1589-1601, 1999,and references therein all of which are incorporated herein byreference). Fusion of HlyAs to proteins and polypeptides has been shownto result in secretion of these fusion proteins utilizing the hemolysinsecretion system (Blight and Holland, Trends Biotechnol. 1994 November;12(11):450-5; Gentschev et al., Behring Inst Mitt. 1994 December;(95):57-66)

The hemolysin protein (HlyA) contains a C-terminal transport signal(HlyAs) which is approximately 50-60 amino acids in length (Hess et al.,Mol Gen Genet. 1990 November; 224(2):201-8; Jarchau et al., Mol GenGenet. 1994 Oct. 17; 245(1):53-60). The HlyA protein is secreted acrossthe inner and outer cellular membranes by the hemolysin secretionsystem. This complex contains three membrane proteins. Two of theseproteins, HlyB and HlyD, are located in the inner membrane, and thethird TolC, is located at the outer membrane. Genes encoding theseproteins are part of the hemolysin operon which consists of four geneshlyC, hlyA, hlyB, and hlyD (Wagner et al., J Bacteriol. 1983 April;154(1):200-10; Gentschev. Gene. 1996 Nov. 7; 179(1):133-40).

In a preferred embodiment for use of the Hly secretion system, DNAplasmids (vectors) are used to express fusion proteins containing theHlyAs signal peptide and allergenic or immunomodulatory polypeptides.The genes encoding the transport complex (hlyB, and hlyD) are encoded bythe same vector. It is recognized that multiple vectors can be used toencode and express these genes, or that sequences encoding these genescan be inserted into the host genome for expression. Preferably, asingle vector contains the complete hemolysin operon including the hlyspecific promoter and an enhancer-type regulator hlyR; the HlyA genewhere only the minimal polypeptide sequence necessary to transport afusion protein is present; and the antigen of interest. TolC protein isgenerally produced by the host E. coli system. However, in systems wheretolC DNA is not encoded by a host organism, tolC can be encoded by avector.

In a particularly preferred embodiment, the secretion plasmid pMOhly1described in WO 98/50067 (“Donner”) is used to express fusion proteinscontaining secretion signal sequences and polypeptides related toinducing anaphylaxis in individuals. The secretion vector pMOhly1contains the complete hemolysin operon including the hly specificpromoter and an enhancer-type regulator hlyR. A majority of the hlyAgene has been deleted so that HlyA encodes only the 34 amino terminaland 61 carboxyl terminal amino acids (HlyA_(s)). A unique Nsirestriction enzyme site between the amino terminal and carboxyl terminalresidues of HlyA facilitates the insertion of heterologous genes or genefragments into the reading frame of HlyA_(s). The genetic informationfor antigens the size of 10-1000 amino acids can be inserted into thissecretion vector pMOhly1, which facilitates the secretion of theseantigens in attenuated Salmonella and other gram-negative attenuatedinoculation strains (e.g. E. coli, Vibrio cholera, Yersinaenterocolitica). In contrast to other secretion systems, the secretionof fusion proteins using a single plasmid is described by Donner. Anadvantage of the hemolysin secretion system in comparison toconventional transport systems is the larger size of the fusion proteinssynthesized and secreted according to the methods taught in Donner.Conventional secretion systems for the presentation of antigens are onlycapable of secreting relatively short peptides to the outer part of thebacterial cell (Cardenas and Clements, Clin Microbiol Rev. 1992 July;5(3):328-42).

Antigens and Allergens

In general, any allergen may be produced by microorganisms in accordancewith the present invention. Preferred allergens are found in certainfoods, venom, drugs or rubber and are capable of eliciting allergicresponses, and in particular anaphylactic allergic responses in anindividual. Particularly preferred allergens are protein or polypeptideallergens.

In a preferred embodiment, microorganisms of the present inventionproduce allergenic proteins that elicit allergies, possibly anaphylaxis,and are found in foods, venoms, drugs, and rubber-based products.Particularly preferred allergenic proteins that induce anaphylaxis, suchas several protein allergens found in food (peanut, milk, egg, wheat),insect venom (i.e. bees, reptiles), drugs, and latex. Non-limitingexamples of protein allergens found in food include proteins found innuts (e.g., peanut walnut, almond, pecan, cashew, hazelnut, pistachio,pine nut, brazil nut), seafood (e.g. shrimp, crab, lobster, clams),fruit (e.g. plums, peaches, nectarines; Ann Allergy Asthma Immunol7(6):504-8 (1996); cherries, Allergy 51(10):756-7 (1996)), seeds(sesame, poppy, mustard), and soy and dairy products (e.g., egg, milk).

Some protein allergens found in nuts are related to legume allergies andmay be used instead of the legume proteins (e.g. peanuts, soybeans,lentils; Ann Allergy Asthma Immunol 77(6): 480-2 (1996). Also, proteinantigens found in pollen-related food allergies may be used (e.g. birchpollen related to apple allergies). Other protein allergens found infoods include those found in young garlic (Allergy 54(6):626-9 (1999),and for children allergic to house dust mites, allergens found in snails(Arch Pediatr 4(8):767-9 (1997)). Protein allergens in wheat are knownto cause exercise-induced allergies (J Allergy Clin Immunol 1999 May;103(5 Pt 1):912-7).

Stings from organisms that inject venoms, such as insect stings areknown to cause anaphylaxis in individuals with allergies to the venom.In general, insect venom includes venom from Hymenoptera such as bees,hornets, wasps, yellow jackets, velvet ants, and fire ants. Inparticular for example, venom from honey bees of the genus Apis cancause anaphylaxis in stung victims who are allergic (Weber et al.Allergy 42:464-470). The venom from honey bees contains numerouscompounds which have been extensively studied and characterized (see fora reference, Banks and Shipolini. Chemistry and Pharmacology ofHoney-bee Venom. Chapter 7 of Venoms of the Hymenoptera. Ed. T. Piek.Academic Press. London. 1986). The two main components of bee venom arephospholipase A2 and melittin and are preferred protein allergens foruse in the present invention for treating and preventing allergies tobee venom.

In certain uses of the present invention, it will be desirable to workin systems in which a single compound (e.g., a single protein) isresponsible for most observed allergy. In other cases, the invention canbe applied to more complex allergens. Therefore, collections of morethan one antigen can be used so that immune responses to multipleantigens may be modulated simultaneously.

Appendix A presents a representative list of certain known proteinantigens. As indicated, the amino acid sequence is known for many or allof these proteins, either through knowledge of the sequence of theircognate genes or through direct knowledge of protein sequence, or both.Of particular interest are anaphylactic antigens.

In another embodiment of allergenic antigens, microorganisms aregenetically engineered to synthesize and secrete modified allergenicpolypeptides that elicit anaphylaxis when exposed to individuals who aresusceptible to anaphylactic shock. Preferably, the allergens aremodified such that the ability to elicit anaphylaxis is reduced oreliminated. As previously discussed allergens elicit allergic responseswhich are sometimes severe enough to induce anaphylactic shock bycrosslinking IgE antibodies bound to the surface of mast cells andbasophils. The IgE crosslinking releases compounds such as histamineswhich causes symptoms related to allergies and anaphylactic shock. Inaccordance with the present invention, microorganisms are used tosynthesize and secrete antigens which are modified to reduce oreliminate IgE binding sites while still maintaining antigenicity orimmunomodulatory activity (U.S. Ser. No. 09/141,220 incorporated hereinby reference). This reduces the risk of allergic or anaphylacticresponses in individuals treated with vaccines containing theseengineered microorganisms.

The amount of antigen to be employed in any particular composition orapplication will depend on the nature of the particular antigen and ofthe application for which it is being used, as will readily beappreciated by those of ordinary skill in the art. The experimentsdescribed in Examples 1-4 suggest that larger amounts of polypeptidesare useful for inducing Th1 responses. The amount of antigen can becontrolled by a variety of factors including but not limited toexpression systems, inducible expression systems, levels of secretionand excretion, methods of killing bacteria before delivery. Those ofordinary skill in the art are capable of determining the desired levelsof antigens to be produced by bacteria and delivered to individuals.

It is recognized that multiple antigenic molecules may be delivered bybacteria simultaneously in accordance with the methods of the presentinvention. Without limitation, different antigenic determinants for oneantigenic protein may be delivered. Different antigenic determinantsfrom different antigenic proteins may also be delivered. Further,multiple antigenic polypeptides and proteins may be delivered inaccordance with the present invention. It is also recognized that singleor multiple antigenic polypeptides and single or multiple cytokines maybe delivered to individuals by bacteria in accordance with the presentinvention. For example but without limitation, allergenic antigens ofthe present invention and immunomodulatory molecules such asinterleukins may be delivered by bacteria using secreted or non-secretedmethods in accordance with the present invention.

Diagnostic and Therapeutic Reagents

The first step in making the modified allergen is to identify IgEbinding sites and/or immunodominant IgE binding sites. The second stepis to mutate one or more of the IgE binding sites, preferably includingat a minimum one of the immunodominant sites, or to react the allergenwith a compound that selectively blocks binding to one or more of theIgE binding sites. The third step is to make sufficient amounts of themodified allergen for administration to persons or animals in need oftolerance to the allergen, where the modified allergen is administeredin a dosage and for a time to induce tolerance, or for diagnosticpurposes. The modified allergen can be administered by injection, or insome cases, by ingestion or inhalation.

A. Allergens.

Many allergens are known that elicit allergic responses, which may rangein severity from mildly irritating to life-threatening. Food allergiesare mediated through the interaction of IgE to specific proteinscontained within the food. Examples of common food allergens includeproteins from peanuts, milk, grains such as wheat and barley, soybeans,eggs, fish, crustaceans, and mollusks. These account for greater than90% of the food allergies (Taylor, Food Techn. 39, 146-152 (1992). TheIgE binding epitopes from the major allergens of cow milk (Ball, et al.(1994) Clin. Exp. Allergy, 24, 758-764), egg (Cooke, S. K. and Sampson,H. R. (1997) J. Immunol., 159, 2026-2032), codfish (Aas, K., andElsayed, S. (1975) Dev. Biol. Stand. 29, 90-98), hazel nut (Elsayed, etal. (1989) Int. Arch. Allergy Appl. Immunol. 89, 410-415), peanut (Burkset al., (1997) Eur. J. Biochemistry, 245:334-339; Stanley et al., (1997)Archives of Biochemistry and Biophysics, 342:244-253), soybean (Herein,et al. (1990) Int. Arch. Allergy Appl. Immunol. 92, 193-198) and shrimp(Shanty, et al. (1993) J. Immunol. 151, 5354-5363) have all beenelucidated, as have others Other allergens include proteins from insectssuch as flea, tick, mite, fire ant, cockroach, and bee as well as molds,dust, grasses, trees, weeds, and proteins from mammals including horses,dogs, cats, etc.

The majority of allergens discussed above elicit a reaction wheningested, inhaled, or injected. Allergens can also elicit a reactionbased solely on contact with the skin. Latex is a well known example.Latex products are manufactured from a milky fluid derived from therubber tree, Hevea brasiliensis and other processing chemicals. A numberof the proteins in latex can cause a range of allergic reactions. Manyproducts contain latex, such as medical supplies and personal protectiveequipment. Three types of reactions can occur in persons sensitive tolatex: irritant contact dermatitis, and immediate systemichypersensitivity. Additionally, the proteins responsible for theallergic reactions can fasten to the powder of latex gloves This powdercan be inhaled, causing exposure through the lungs. Proteins found inlatex that interact with IgE antibodies were characterized bytwo-dimensional electrophoresis. Protein fractions of 56, 45, 30, 20,−14, and less than 6.5 kd were detected (Posch A. et al., (1997) J.Allergy Clin. Immunol. 99(3), 385-395). Acidic proteins in the 8-14 kdand 22-24 kd range that reacted with IgE antibodies were also identified(Posch A. et al., (1997) J. Allergy Clin. Immunol. 99(3), 385-395. Theproteins prohevein and hevein, from hevea brasiliensis, are known to bemajor latex allergens and to interact with IgE (Alenius, H., et al.,Clin. Exp. Allergy 25(7), 659-665; Chen Z., et al., (1997). J. AllergyClin. Immunol. 99(3), 402-409). Most of the IgE binding domains havebeen shown to be in the hevein domain rather than the domain specificfor prohevein (Chen Z., et al., (1997) J. Allergy Clin. Immunol. 99(3),402-409). The main IgE-binding epitope of prohevein is thought to be inthe N-terminal, 43 amino acid fragment (Alenius H., et al., (1996) J.Immunol. 156(4), 1618-1625). The hevein lectin family of proteins hasbeen shown to have homology with potato lectin and snake venomdisintegrins (platelet aggregation inhibitors) (Kielisqewski, M. L., etal., (1994) Plant J. 5(6), 849-861).

B. Identification of IgE Binding Sites.

Allergens typically have both IgE and IgG binding sites and arerecognized by T cells. The binding sites can be determined either byusing phage display libraries to identify conformational epitopes(Eichler and Houghten, (1995) Molecular Medicine Today 1, 174-180;Jensen-Jarolim et al., (1997) J. Appl. Clin. Immunol. 101, 5153a) or byusing defined peptides derived from the known amino acid sequence of anallergen (see examples below), or by binding of whole protein or proteinfragments to antibodies, typically antibodies obtained from a pooledpatient population known to be allergic to the allergen. It is desirableto modify allergens to diminish binding to IgE while retaining theirability to activate T cells and in some embodiments by not significantlyaltering or decreasing IgG binding capacity. This requires modificationof one or more IgE binding sites in the allergen.

A preferred modified allergen is one that can be used with a majority ofpatients having a particular allergy. Use of pooled sera from allergicpatients allows determination of one or more immunodominant epitopes inthe allergen. Once some or all of the IgE binding sites are known, it ispossible to modify the gene encoding the allergen, using site directedmutagenesis by any of a number of techniques, to produce a modifiedallergen as described below, and thereby express modified allergens. Itis also possible to react the allergen with a compound that achieves thesame result as the selective mutation, by making the IgE binding sitesinaccessible, but not preventing the modified allergen from activating Tcells, and, in some embodiments, by not significantly altering ordecreasing IgG binding.

Assays to assess an immunologic change after the administration of themodified allergen are known to those skilled in the art. Conventionalassays include RAST (Sampson and Albergo, 1984), ELISAs (Burks, et al.1986) immunoblotting (Burks, et al. 1988), and in vivo skin tests(Sampson and Albergo 1984). Objective clinical symptoms can be monitoredbefore and after the administration of the modified allergen todetermine any change in the clinical symptoms.

It may be of value to identify IgEs which interact with conformationalrather than linear epitopes. Due to the complexity and heterogeneity ofpatient serum, it may be difficult to employ a standard immobilizedallergen affinity-based approach to directly isolate these IgEs inquantities sufficient to permit their characterization. These problemscan be avoided by isolating some or all of the IgEs which interact withconformational epitopes from a combinatorial IgE phage display library.

Steinberger et al. (Steinberger, P., Kraft D. and Valenta R. (1996)“Construction of a combinatorial IgE library from an allergic patient:Isolation and characterization of human IgE Fabs with specificity forthe major Timothy Grass pollen antigen,” Ph1 p. 5 J. Biol. Chem. 271,10967-10972) prepared a combinatorial IgE phage display library frommRNA isolated from the peripheral blood mononuclear cells of a grassallergic patient. Allergen-specific IgEs were selected by panningfilamentous phage expressing IgE Fabs on their surfaces against allergenimmobilized on the wells of 96 well microtiter plates. The cDNAs werethan isolated from allergen-binding phage and transformed into E. colifor the production of large quantities of monoclonal, recombinant,allergen-specific IgE Fabs.

If native allergen or full length recombinant allergen is used in thepanning step to isolate phage, then Fabs corresponding to IgEs specificfor conformational epitopes should be included among theallergen-specific clones identified. By screening the individualrecombinant IgE Fabs against denatured antigen or against the relevantlinear epitopes identified for a given antigen, the subset ofconformation-specific clones which do not bind to linear epitopes can bedefined.

To determine whether the library screening has yielded a completeinventory of the allergen-specific IgEs present in patient serum, animmunocompetition assay can be performed. Pooled recombinant Fabs wouldbe preincubated with immobilized allergen. After washing to removeunbound Fab, the immobilized allergen would then be incubated withpatient serum. After washing to remove unbound serum proteins, anincubation with a reporter-coupled secondary antibody specific for IgEFc domain would be performed. Detection of bound reporter would allowquantitation of the extent to which serum IgE was prevented from bindingto allergen by recombinant Fab. Maximal, uncompeted serum IgE bindingwould be determined using allergen which had not been preincubated withFab or had been incubated with nonsense Fab. If IgE binding persists inthe face of competition from the complete set of allergen-specific IgEFab clones, this experiment can be repeated using denatured antigen todetermine whether the epitopes not represented among the cloned Fabs arelinear or conformational.

Production of Recombinant or Modified Allergens

A modified allergen will typically be made using recombinant techniques.Expression in a procaryotic or eucaryotic host including bacteria,yeast, and baculovirus-insect cell systems are typically used to producelarge (mg) quantities of the modified allergen. It is also possible tomake the allergen synthetically, if the allergen is not too large, forexample, less than about 25-40 amino acids in length.

Adjuvants and Immunostimulatory Agents

Compositions and methods of the present invention include the use ofadjuvants and immunomodulatory polypeptides or immunostimulatory factorsto modulate an individual's immune response. Immunologic adjuvants areagents that enhance specific immune responses to vaccines. Formulationof vaccines with potent adjuvants is desirable for improving theperformance of vaccines composed of antigens. Adjuvants may have diversemechanisms of action and should be selected for use based on the routeof administration and the type of immune response (antibody,cell-mediated, or mucosal immunity) that is desired for a particularvaccine

In general, immunomodulatory polypeptides include cytokines which aresmall proteins or biological factors (in the range of 5-20 kD) that arereleased by cells and have specific effects on cell-cell interaction,communication and behavior of other cells. As previously described,cytokines in accordance with the present invention are proteins that aresecreted to T-cells to induce a Th1 or Th2 response. Preferably, thecytokine(s) to be administered is/are selected to reduce production of aTh2 response to antigens associated with anaphylaxis. One preferredmethod of reducing a Th2 response is through induction of thealternative response. Cytokines that, when expressed during antigendelivery into cells, induce a Th1 response in T cells (i.e., “Th1stimulating cytokines”) include IL-12, IL-2, I-18, IL-1 or fragmentsthereof, IFN, and/or IFNγ.

Other compounds that are immunomodulatory include immunological inducingagents. These inducing agents prompt the expression of Th1 stimulatingcytokines by T-cells and include factors such as, CD40, CD40 ligand,oligonucleotides containing CpG motifs, TNF, and microbial extracts suchas preparations of Staphylococcus aureus, heat killed Listeria, andmodified cholera toxin, etc.

Those of ordinary skill in the art readily appreciate the preferredtypes of adjuvants for use with particular antigen compositions. Ingeneral, immunologic adjuvant include gel-type adjuvants (e.g. aluminumhydroxide/aluminum phosphate, calcium phosphate), microbial adjuvants(e.g. DNA such as CpG motifs; endotoxin such as monophosphoryl lipid A;exotoxins such as cholera toxin, E. coli heat labile toxin, andpertussis toxin; and muramyl dipeptide), oil-emulsion andemulsifier-based adjuvants (e.g. Freund's Incomplete Adjuvant, MF59, andSAF), particulate adjuvants (e.g. liposomes, biodegradable microspheres,and saponins), and synthetic adjuvants (e.g. nonionic block copolymers,muramyl peptide analogues, polyphosphazene, and syntheticpolynucleotides).

Adjuvants that are known to stimulate Th2 responses are preferablyavoided. Particularly preferred adjuvants include, for example,preparations (including heat-killed samples, extracts, partiallypurified isolates, or any other preparation of a microorganism ormacroorganism component sufficient to display adjuvant activity) ofmicroorganisms such as Listeria monocytogenes or others (e.g., BacilleCalmette-Guerin [BCG], Corynebacterium species, Mycobacterium species,Rhodococcus species, Eubacteria species, Bortadella species, andNocardia species), and preparations of nucleic acids that includeunmethylated CpG motifs (see, for example, U.S. Pat. No. 5,830,877; andpublished PCT applications WO 96/02555, WO 98/18810, WO 98/16247, and WO98/40100, each of which is incorporated herein by reference). Otherpreferred adjuvants reported to induce Th1-type responses and notTh2-type responses include, for example, Aviridine(N,N-dioctadecyl-N′N′-bis(2-hydroxyethyl)propanediamine) and CRL 1005.Particularly preferred are ones that induce IL-12 production, includingmicrobial extracts such as fixed Staphylococcus aureus, Streptococcalpreparations, Mycobacterium tuberculosis, lipopolysaccharide (LPS),monophosphoryl lipid A (MPLA) from gram negative bacteriallipopolysaccharides (Richards et al. Infect Immun 1998 June;66(6):2859-65), listeria monocytogenes, toxoplasma gondii, leishmaniamajor. Some polymers are also adjuvants. For example, polyphosphazenesare described in U.S. Pat. No. 5,500,161 to Andriavnov, et al. These canbe used not only to encapsulate the microorganisms but also to enhancethe immune response to the antigen.

If adjuvants are not synthesized by microorganisms in accordance withthe present invention, adjuvants which are cytokines may be provided asimpure preparations (e.g., isolates of cells expressing a cytokine gene,either endogenous or exogenous to the cell), but are preferably providedin purified form. Purified preparations are preferably at least about90% pure, more preferably at least about 95% pure, and most preferablyat least about 99% pure. Alternatively, genes encoding the cytokines orimmunological inducing agents may be provided, so that gene expressionresults in cytokine or immunological inducing agent production either inthe individual being treated or in another expression system (e.g., anin vitro transcription/translation system or a host cell) from whichexpressed cytokine or immunological inducing agent can be obtained foradministration to the individual. It is recognized that microorganismsutilized to synthesize and deliver allergenic and/or immunomodulatoryproteins according to the present invention can act as an adjuvant, andthat preferred microorganisms are immunostimulatory adjuvants.

It will be appreciated by those of ordinary skill in the art that theinventive administration of microorganisms expressing cytokines and/orallergens may optionally be combined with the administration of anyother desired immune system modulatory factor such as, for example, anadjuvant or other immunomodulatory compound.

Methods of Administration

Formulations can be delivered to a patient by any available routeincluding for example enteral, parenteral, topical (including nasal,pulmonary or other mucosal route), oral or local administration. Thecompositions are preferably administered in an amount effective toelicit cellular immunity and production of Th1-related IgG whileminimizing IgE mediated responses. Also preferred are compositionsadministered in an effective amount to active T-cell response,preferably Th1-type responses. For compositions of the present inventioncontaining bacteria, administration is preferably deliveredparenterally.

It is important to administer the modified allergen to an individual(human or animal) to decrease the clinical symptoms of allergic diseaseby using a method, dosage, and carrier which are effective. Allergenwill typically be administered in an appropriate carrier, such as salineor a phosphate saline buffer. Allergen can be administered by injectionsubcutaneously, intramuscularly, or intraperitoneally (most humans wouldbe treated by subcutaneous injection), by aerosol, inhaled powder, or byingestion.

Pharmaceutical Compositions

Pharmaceutical compositions for use in accordance with the presentinvention may include a pharmaceutically acceptable excipient orcarrier. As used herein, the term “pharmaceutically acceptable carrier”means a non-toxic, inert solid, semi-solid or liquid filler, diluent,encapsulating material or formulation auxiliary of any type. Someexamples of materials which can serve as pharmaceutically acceptablecarriers are sugars such as lactose, glucose, and sucrose; starches suchas corn starch and potato starch; cellulose and its derivatives such assodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate;powdered tragacanth; malt; gelatin; talc; excipients such as cocoabutter and suppository waxes; oils such as peanut oil, cottonseed oil;safflower oil; sesame oil; olive oil; corn oil and soybean oil; glycols;such as propylene glycol; esters such as ethyl oleate and ethyl laurate;agar; buffering agents such as magnesium hydroxide and aluminumhydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer'ssolution; ethyl alcohol, and phosphate buffer solutions, as well asother non-toxic compatible lubricants such as sodium lauryl sulfate andmagnesium stearate, as well as coloring agents, releasing agents,coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the composition,according to the judgment of the formulator. The pharmaceuticalcompositions of this invention can be administered to humans and/or toother animals, orally, rectally, parenterally, intracisternally,intravaginally, intraperitoneally, topically (as by powders, ointments,or drops), bucally, or as an oral or nasal spray.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups andelixirs. In addition to the active compounds, the liquid dosage formsmay contain inert diluents commonly used in the art such as, forexample, water or other solvents, solubilizing agents and emulsifierssuch as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, dimethylformamide, oils (in particular, cottonseed, groundnut,corn, germ, olive, castor, and sesame oils), glycerol,tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid estersof sorbitan, and mixtures thereof. Besides inert diluents, the oralcompositions can also include agents such as wetting agents, emulsifyingand suspending agents, sweetening, flavoring, and perfuming agents.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

In order to prolong the effect of an agent, it is often desirable toslow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material with poor water solubility. The rateof absorption of the agent then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle. Injectable depot forms are made by forming microencapsulematrices of the drug in biodegradable polymers such aspolylactide-polyglycolide. Depending upon the ratio of agent to polymerand the nature of the particular polymer employed, the rate of releaseof the agent can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides) Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissues.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the compounds of thisinvention with suitable non-irritating excipients or carriers such ascocoa butter, polyethylene glycol or a suppository wax which are solidat ambient temperature but liquid at body temperature and therefore meltin the rectum or vaginal cavity and release the active compound.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like.

The solid dosage forms of tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions which can beused include polymeric substances and waxes.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like.

The compounds can also be in micro-encapsulated form with one or moreexcipients as noted above. The solid dosage forms of tablets, dragees,capsules, pills and granules can be prepared with coatings and shellssuch as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art. In such soliddosage forms the active compound may be admixed with at least one inertdiluent such as sucrose, lactose or starch. Such dosage forms may alsocomprise, as is normal practice, additional substances other than inertdiluents, e.g., tableting lubricants and other tableting aids such amagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets and pills, the dosage forms may also comprisebuffering agents. They may optionally contain opacifying agents and canalso be of a composition that they release the active ingredient(s)only, or preferentially, in a certain part of the intestinal tract,optionally, in a delayed manner. Examples of embedding compositionswhich can be used include polymeric substances and waxes.

Dosage forms for topical or transdermal administration of an inventivepharmaceutical composition include ointments, pastes, creams, lotions,gels, powders, solutions, sprays, inhalants or patches. The activecomponent is admixed under sterile conditions with a pharmaceuticallyacceptable carrier and any needed preservatives or buffers as may berequired. Ophthalmic formulation, ear drops, eye drops are alsocontemplated as being within the scope of this invention.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of this invention, excipients such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to the compounds of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants suchas chlorofluorohydrocarbons.

Transdermal patches have the added advantage of providing controlleddelivery of a compound to the body. Such dosage forms can be made bydissolving or dispensing the compound in the proper medium. Absorptionenhancers can also be used to increase the flux of the compound acrossthe skin. The rate can be controlled by either providing a ratecontrolling membrane or by dispersing the compound in a polymer matrixor gel.

Encapsulation

In a preferred embodiment, inventive compositions comprising livemicroorganisms are provided in association with an encapsulation device(see, for example, U.S. Ser. No. 60/169,330 entitled “ControlledDelivery of Antigens” filed Dec. 6, 1999, incorporated by referenceherewith). Preferred encapsulation devices are biocompatible, are stableinside the body so that microorganisms are not released until after theencapsulation device reaches its intended destination (e.g. mucosallining of the gut, endocytosis by antigen-presenting cells (APC)). Forexample, preferred systems of encapsulation are stable at physiologicalpH and degrade at acidic pH levels comparable to those found in thedigestive tract or endosomes of APCs. Particularly preferredencapsulation compositions include but are not limited to onescontaining liposomes, polylactide-co-glycolide (PLGA), chitosan,synthetic biodegradable polymers, environmentally responsive hydrogels,and gelatin PLGA nanoparticles. Inventive compositions may beencapsulated in combination with one or more adjuvants, targetingentities, or other agents including, for example, pharmaceuticalcarriers, diluents, excipients, oils, etc. Alternatively or additionallythe encapsulation device itself may be associated with a targetingentity and/or an adjuvant.

Methods of encapsulating live cells are known and may also be used inaccordance with the present invention for delivering antigen-secretingmicroorganisms to individuals. The following references are provided asexamples of encapsulation of live cells. However, any method ofencapsulating live cells may be used in the present invention. U.S. Pat.No. 5,084,350; U.S. Pat. No. 4,680,174; and U.S. Pat. No. 4,352,883 (allof which are incorporated herein by reference) describe theencapsulation of a prokaryotic or eukaryotic cell or cell culture inmicrocapsules. Briefly, U.S. Pat. Nos. 5,084,350; 4,680,174; and4,352,883 disclose that a tissue sample, cell, or cell culture to beencapsulated is first prepared in finely divided form in accordance withwell-known techniques and suspended in an aqueous medium suitable formaintenance and for supporting the ongoing metabolic processes of theparticular cells involved. Media suitable for this purpose generally areavailable commercially. Thereafter, a water-soluble substance which isphysiologically compatible with the cells and which can be renderedwater-insoluble to form a shape-retaining coherent spheroidal mass orother shape is added to the medium. The solution is then formed intodroplets containing cells together with their maintenance or growthmedium and is immediately rendered water-insoluble and gelled to formshape-retaining, typically spheroidal coherent masses.

The material used to induce gelation of the culture medium may be anynon-toxic water-soluble material which, by a change in the surroundingtemperature, pH, ionic environment, or concentration, can be convertedto shape-retaining masses. Preferably, the material also is one whichcomprises plural, easily ionized groups, e.g., carboxyl or amino groups,which can react by salt formation with polymers containing plural groupswhich ionize to form species of the opposite charge. Use of this type ofmaterial enables the deposition of a membrane of a selected porosityrange without damage to the labile cells. The presently preferredmaterials for forming the gelled masses are water-soluble natural orsynthetic polysaccharides. Many such commercially available materialsare typically extracted from vegetable matter and are often used asadditives in various foods. Sodium alginate is the presently preferredwater-soluble polysaccharide. Other usable materials include acidicfractions of guar gum, gum arabic, carrageenan, pectin, tragacanth gumor xanthan gums. These materials may be gelled when multivalent ions areexchanged for the acidic hydrogen or alkali metal ion normallyassociated with the carboxyl groups.

Uses

The compositions of the present invention may be employed to treat orprevent allergic reactions in a subject. Subjects are animal and humanpatients in need of treatment for allergies. Preferably, the animal is adomesticated mammal (e.g., a dog, a cat, a horse, a sheep, a pig, agoat, a cow, etc). Animals also include laboratory animals such as mice,rats, hamsters, monkeys, and rabbits. Any individual who suffers fromallergy, or who is susceptible to allergy, may be treated. It will beappreciated that an individual can be considered susceptible to allergywithout having suffered an allergic reaction to the particular antigenin question. For example, if the individual has suffered an allergicreaction to a related antigen (e.g., one from the same source or one forwhich shared allergies are common), that individual will be consideredsusceptible to allergy to the relevant antigen. Similarly, if members ofan individual's family are allergic to a particular antigen, theindividual may be considered to be susceptible to allergy to thatantigen. More preferably, any individual who is susceptible toanaphylactic shock upon exposure to food allergens, venom allergens orrubber allergens may be treated according to the present invention.

The compositions of the present invention may be formulated for deliveryby any route. Preferably, the compositions are formulated for injection,ingestion, or inhalation.

Therapy or desensitization with the modified allergens can be used incombination with other therapies, such as allergen-non-specific anti-IgEantibodies to deplete the patient of allergen-specific IgE antibodies(Boulet, et al. (1997) 155:1835-1840; Fahy, et al. (1997) American JRespir. Crit. Care Med. 155:1828-1834; Demoly, P. and Bousquet (1997) JAm J Resp. Crit. Care Med. 155:1825-1827), or by the pan specificanti-allergy therapy described in U.S. Ser. No. 08/090,375 filed Jun. 4,1998, by M. Caplan and H. Sosin. Therapy with the modified allergen canalso be administered in combination with an adjuvant such as IL-12,IL-16, IL-18, IFNγ.

The nucleotide molecule encoding the modified allergen can also beadministered directly to the patient, for example, in a suitableexpression vector such as a plasmid, which is injected directly into themuscle or dermis, or through administration of genetically engineeredcells.

In general, effective dosages will be in the picogram to milligramrange, more typically microgram to milligram. Treatment will typicallybe between twice/weekly and once a month, continuing for up to three tofive years, although this is highly dependent on the individual patientresponse.

The modified allergen can also be used as a diagnostic to characterizethe patient's allergies, using techniques such as those described in theexamples.

Modifications and variations of the methods and compositions describedherein are intended to be within the scope of the following claims.

Other Embodiments

Those of ordinary skill in the art will readily appreciate that theforegoing represents merely certain preferred embodiments of theinvention. Various changes and modifications to the procedures andcompositions described above can be made without departing from thespirit or scope of the present invention, as set forth in the followingclaims.

EXAMPLES Material and Methods

For general methods used to express proteins in microorganisms seeAusubel et al. (supra) and Sambrook et al. (supra) both of which areincorporated herein by reference. In addition, expression vectors foruse in the present invention are widely available from commercialsources (see for example, Clontech, Palo Alto, Calif.; Invitrogen,Carlsbad, Calif.; Promega Corporation, Madison, Wis.; New EnglandBiolabs, Beverly, Mass.).

The following experiments describe the encapsulation of allergens inbacteria for use as a delivery vehicle and/or adjuvant in immunotherapyin accordance with the teachings of the present invention. Recombinantpeanut allergen proteins (Ara h 1, Ara h 2, and Ara h 3; Burks et al. JAllergy Clin Immunol. 88(2):172-9, 1991; Burks et al. J Allergy ClinImmunol. 90(6 Pt 1):962-9, 1992; Rabjohn et al. J Clin Invest.103(4):535-42, 1999; incorporated herein by reference) were produced inE. coli BL21 cells by transforming the bacterial cells with cDNA clonesencoding the proteins (see Appendix B; sequences cloned into pET24,Novagen, Madison, Wis.). The transformed cells were then injected intoC3H/HEJ mice to determine if the allergen-expressing E. coli elicited animmune response.

Example 1 Methods of Killing Allergen-Producing E. Coli

Several methods of killing allergen-producing E. coli were tested.Preferably, the method of killing bacteria does not denature orproteolyze the recombinant allergen(s) produced by the bacteria. Asnon-limiting examples, E. coli were killed by heat (at temperaturesranging from 37° C. to 95° C.), by using ethanol (0.1% to 10%), and byusing solutions containing iodine (0.1% to 10%). Survival was determinedby plating 100 μl of cells onto the appropriate agar plates, andsubsequently counting the resulting colonies. The most reproduciblemethod was heat killing. Therefore, the preferred method of killingallergen-producing E. coli is to incubate the cells at 60° C. for 20minutes which results in 100% death (i.e. no colonies formed; see FIG.1).

Example 2 Growth of Bacteria

The following protocol was developed for the preparation ofallergen-producing E. coli cells for inoculation of mice.

Day 1

Five milliliters (ml) of liquid cultures of LB (Luria-Bertani broth)containing kanamycin (30 micrograms/ml per each cell line used) wereprepared in 50 ml sterile tubes or flasks. Cultures were inoculated withapproximately 10 microliters from a frozen stock of the desiredbacterial cell line containing the desired expression vectors. Theinoculated cultures were incubated with shaking overnight at 37° C.

Day 2

The following morning, 100 ml of liquid LB (500 ml Erlenmeyer flask)containing kanamycin (30 micrograms/ml) were inoculated using a 1 mlaliquot from the 5 ml culture grown from the previous day. (Theremaining 4 mls of culture were frozen. Optionally, the remaining 4milliliters of culture can be stored at 4° C. for several weeks forinoculating subsequent cultures) The inoculated cultures were incubatewith shaking at 37° C. until the optical density of the solutionmeasured at 600 nM (OD₆₀₀) reached approximately 0.6 to 0.9.

Day 3

To induce production of recombinant proteins, the cultures from theprevious day were induced by addingisopropyl-beta-D-thiogalactopyranoside (IPTG; Sigma-Aldrich, St. Louis,Mo.) from a 1 M stock to a final concentration of 1 mM (100 microlitersof 1 M IPTG per 100 mls of culture) when the OD₆₀₀ of the culturereached approximately 0.6-0.9. The induced cultures were incubatedovernight.

Day 4

1.4 ml of culture from the previous day were aliquoted into each of five1.5 ml microfuge tubes for each culture and heat killed at 60° C. in awater bath for 20 minutes. The tubes were centrifuged at 16,000×g for 5minutes at room temperature and the supernatant discarded. The pelletswere washed with 1× phosphate buffer saline (PBS) and centrifuged at16,000×g for 5 minutes at room temperature. Again, the supernatant wasdiscarded and the pellets were resuspended in 250 microliters of 1×PBS.The resuspended pellets from the same original samples were combined.The OD₆₀₀ were determined for each sample and diluted to the desiredOD₆₀₀ using 1×PBS.

Example 3 Production and Release of Allergen Release of Allergen byHeat-Killed Bacteria

In order to determine if the cells remained intact after heat-killing wemeasured the amount of allergen released into the media. A dot-blotassay was developed that utilized as controls, purified recombinantallergens applied to a filter at known concentrations and serum IgE frompeanut sensitive patients. The assay detected and quantified the amountof allergen present in 100 microliters of supernatant after pelletingheat-killed bacteria. The level of allergen released varied and wasdependent on the expression vector and protein tested. In general, moreAra h 2 was released than Ara h 1 and Ara h 3 (Ara h 2>>Ara h 1>Ara h3).

Production of Allergen.

In order to measure amounts of allergen in E. coli, we developed animmunoblot assay that utilizes a six histidine tag (HIS tag) that ispresent on all of our purified recombinant allergens and a HIS tagantibody to build a standard curve that could then be used to estimateamounts of allergen produced. The amount of allergen produced on a percell basis varied depending on which clone was tested. In general, moreAra h 3 was produced than Ara h 2 and Ara h 1 (Ara h 3>Ara h 2>>Ara h1).

Our best estimates for amounts of allergen delivered in 100 μl of a 2.0O.D. inoculum of E. coli varies from about 1 μg of Ara h 1 to about 20μg of Ara h 3.

FIG. 2 is an example of a standard curve generated for Ara h 2. Theoptical density (O.D.) of the HIS-tagged Ara h 2 allergen is thendetermined from an immunoblot where different concentrations of E. coliextract has been electrophoresed on SDS-PAGE gels. The allergen O.D. isthen used to estimate the amount of protein produced by that extract.

Example 4 Immune Response of Mice

The following protocol was utilized to determine the immune response ofmice injected with allergen-producing bacteria. Blood was collected fromthe tail vein of each mouse used before the first injection. Enoughblood was collected for antibody ELISA for each allergen and E. coliproteins. On Day Zero each mouse was injected with 100 microliters ofthe killed E. coli samples subcutaneously in the left hind flank. Themice were injected for the second time on Day 14 using the sameprocedure as Day Zero. On Day 21, a second blood sample was collectedfrom each mouse. Blood samples at Day 0 and Day 21 were assayed for IgG1and IgG2a antibodies to either Ara h 1, Ara h 2, or Ara h 3 by an ELISAassay.

Mice injected with E. coli producing Ara h 1 did not give detectablelevels of any immunoglobulin to the Ara h 1 allergen and therefore, thatdata are not shown. Without limitation to theory, we speculate that thismay be due to the relatively small amounts of Ara h 1 produced by thesecells (see previous discussion). Mice injected with E. coli producingAra h 2 contained relatively high levels of IgG1 and IgG2a. Again,without limitation to the cause, we speculated that this may be due tothe amount of Ara h 2 released from these cells (see discussion above).Mice injected with E. coli producing Ara h 3 contained relatively highlevels of IgG2a (indicative of a Th1-type response) and elicitedrelatively low levels of IgG1 (indicative of a Th2-type response.

Interpretation of Results

The present data should be cautiously interpreted. The data in theFigures only represent O.D. levels and do not represent absolute amountsof immunoglobulin. Therefore comparisons between groups should take intoconsideration the data presented as O.D. However, the general trendsuggests that for example, more mice exhibited an IgG2a response to Arah 3 than mice that exhibit an IgG1 response to Ara h 3.

Examples 5-9

Peanut allergy is one of the most common and serious of the immediatehypersensitivity reactions to foods in terms of persistence and severityof reaction. Unlike the clinical symptoms of many other food allergies,the reactions to peanuts are rarely outgrown, therefore, most diagnosedchildren will have the disease for a lifetime (Sampson, H. A., andBurks, A. W. (1996) Annu. Rev. Nutr. 16, 161-77; Bock, S. A. (1985). J.Pediatr. 107, 676-680). The majority of cases of fatal food-inducedanaphylaxis involve ingestion of peanuts (Sampson et al., (1992) NEJM327, 380-384; Kaminogawa, S. (1996) Biosci. Biotech. Biochem. 60,1749-1756). The only effective therapeutic option currently availablefor the prevention of a peanut hypersensitivity reaction is foodavoidance. Unfortunately, for a ubiquitous food such as a peanut, thepossibility of an inadvertent ingestion is great.

The examples described below demonstrate identification, modification,and assessment of allergenicity of the major peanut allergens, Ara h 1,Ara h 2, and Ara h 3. Detailed experimental procedures are included forExample 1. These same procedures were used for Examples 2-5. Thenucleotide sequences of Ara h 1, Ara h 2, and Ara h 3, are shown in SEQID NOs. 1, 3, and 5, respectively. The amino acid sequences of Ara h 1,Ara h 2, and Ara h 3 are shown in SEQ ID NOs. 2, 4, and 6 respectively.

Example 5 Identification of Linear IgE Binding Epitopes

Due to the significance of the allergic reaction and the widening use ofpeanuts as protein extenders in processed foods, there is increasinginterest in defining the allergenic proteins and exploring ways todecrease the risk to the peanut-sensitive individual. Various studiesover the last several years have identified the major allergens inpeanuts as belonging to different families of seed storage proteins(Burks, et al. (1997) Eur. J. Biochem. 245, 334-339; Stanley, et al.(1997) Arch. Biochem. Biophys. 342, 244-253). The major peanut allergensAra h 1, Ara h 2, and Ara h 3 belong to the vicilin, conglutin andglycinin families of seed storage proteins, respectively. Theseallergens are abundant proteins found in peanuts and are recognized byserum IgE from greater than 95% of peanut sensitive individuals,indicating that they are the major allergens involved in the clinicaletiology of this disease (Burks, et al. (1995). J. Clinical Invest., 96,1715-1721). The genes encoding Ara h 1 (SEQ ID NO. 1), Ara h 2 (SEQ IDNO. 3), and Ara h 3 (SEQ ID NO. 5) and the proteins encoded by thesegenes (SEQ ID NOs. 2, 4, 6) have been isolated and characterized. Thefollowing studies were conducted to identify the IgE epitopes of theseallergens recognized by a population of peanut hypersensitive patientsand a means for modifying their affinity for IgE.

Experimental Procedures

Serum IgE.

Serum from 15 patients with documented peanut hypersensitivity reactions(mean age, 25 yrs) was used to determine relative binding affinitiesbetween wild-type and mutant Ara h 1 synthesized epitopes. The patientshad either a positive double-blind, placebo-controlled, food challengeor a convincing history of peanut anaphylaxis (laryngeal edema, severewheezing, and/or hypotension; Burks, et al. (1988) J. Pediatr. 113,447-451). At least 5 ml of venous blood was drawn from each patient,allowed to clot, and serum was collected A serum pool from 12 to 15patients was made by mixing equal aliquots of serum IgE from eachpatient. The pools were then used in immunoblot analysis.

Peptide Synthesis.

Individual peptides were synthesized on a derivatized cellulose membraneusing 9-fluorenyllmethoxycarbonyl (Fmoc) amino acid active estersaccording to the manufacturer's instructions (Geriosys Biotechnologies,Woodlands, Tex.; Fields, G. B and Noble, R. L. (1990) Int. J. PeptideProtein Res. 35, 161-214). Fmoc-amino acids (N-terminal blocked) withprotected side chains were coupled in the presence of1-methyl-2-pyrrolidone to a derivatized cellulose membrane. Followingwashing with dimethylformamide (DMF), unreacted terminal amino groupswere blocked from further reactions by acetylation with aceticanhydride. The N-terminal Fmoc blocking group was then removed byreaction with 20% piperidine and 80% DMF, v/v. The membrane was washedin DMF followed by methanol, the next reactive Fmoc-amino acid was thencoupled as before, and the sequence of reactions was repeated with thenext amino acid. When peptide synthesis was complete, the side chainswere deprotected with a mixture of dichloromethane (DCM),trifluoroacetic acid, and triisobutylsilane (1.0:1.0:0.5), followed bysuccessive washes in DCM, DMF, and methanol. Peptides synthesisreactions were monitored by bromophenol blue color reactions duringcertain steps of synthesis. Cellulose derivitised membranes andFmoc-amino acids were supplied by Genosys Biotechnologies. All otherchemical were purchased from Aldrich Chemical Company, Inc. (Milwaukee,Wis.) or Fluka (Bucks, Switzerland). Membranes were either probedimmediately or stored at −20° C. until needed.

IgE Binding Assays.

Cellulose membranes containing synthesized peptides were washed 3 timesin Tris-buffered saline (TBS; 136 mM NaCl, 2.7 mM KCl, and 50 mM trizmabase pH 8.0) for 10 min at room temperature (RT) and then incubatedovernight in blocking buffer: [TBS, 0.05% Tween™ 20; concentratedmembrane blocking buffer supplied by Genosys; and sucrose(0.0:1.0:0.5)]. The membrane was then incubated in pooled sera dilutedin 1:5 in 20 mM Tris-Cl pH7.5, 150 mM NaCl, and 1% bovine serum albuminovernight at 4° C. Primary antibody was detected with ¹²⁵I-labeledequine anti-human IgE (Kallestad, Chaska, Minn.).

Quantitation of IgE Binding.

Relative amounts of IgE binding to individual peptides were determinedby a Bio-Rad (Hercules, Calif.) model GS-700 imaging laser densitometerand quantitated with Bio-Rad molecular analyst software. A backgroundarea was scanned and subtracted from the obtained values. Followingquantitation, wild-type intensities were normalized to a value of oneand the mutants were calculated as percentages relative to thewild-type.

Synthesis and Purification of Recombinant Ara h 2 Protein.

cDNA encoding Ara h 2 was placed in the pET-24b expression vector. ThepET-24 expression vector places a 6× histidine tag at the carboxyl endof the inserted protein. The histidine tag allows the recombinantprotein to be purified by affinity purification on a nickel column(HisBind resin). Recombinant Ara h 2 was expressed and purifiedaccording to the instructions of the pET system manual. Briefly,expression of the recombinant Ara h 2 was induced in 200 ml cultures ofstrain BL21(DE3) E. coli with 1 mM IPTG at mid log phase. Cultures wereallowed to continue for an additional 3 hours at 36° C. Cells wereharvested by centrifugation at 2000×g for 15 minutes and then lysed indenaturing binding buffer (6 M urea, 5 mM imidazole, 0.5 M NaCl, 20 mMTris-HCl, pH 7.9). Lysates were cleared by centrifugation at 39,000×gfor 20 minutes followed by filtration though 0.45 micron filters. Thecleared lysate was applied to a 10 ml column of HisBind resin, washedwith imidazole wash buffer (20 mM imidazole, 6 M urea, 0.5 M NaCl, 20 mMTris-HCl, pH 7.9). The recombinant Ara h 2 was then released from thecolumn using elution buffer (1 M imidazole, 0.5 M NaCl, 20 mM Tris-HCl,pH 7.9). The elution buffer was replaced with phosphate buffered salineby dialysis. The purification of recombinant Ara h 2 was followed bySIDS PAGE and immunoblots. Peanut specific serum IgE was used as aprimary antibody.

Skin Prick Tests.

The ability of purified native and recombinant Ara h 2 to elicit the IgEmediated degranulation of mast cells was evaluated using prick skintests in a peanut allergic individual. An individual meeting thecriteria for peanut allergy (convincing history or positive double blindplacebo controlled food challenge) and a non-allergic control wereselected for the testing. Purified native and recombinant Ara h 2 andwhole peanut extract (Greer Laboratories, Lenoir, N.C.) were tested.Twenty microliters of the test solution were applied to the forearm ofthe volunteer and the skin beneath pricked with a sterile needle.Testing was started at the lowest concentration (less than or equal to 1mg/ml) and increased ten fold each round to the highest concentration oruntil a positive reaction was observed. Mean diameters of the wheal anderythema were measured and compared to the negative saline control. Apositive reaction was defined as a wheal 3 mm larger then the negativecontrol. Histamine was used as the positive control.

Results

Identification of the Linear IgE-Binding Epitopes of Ara h 1, Ara h 2and Ara h 3 Allergens.

Epitope mapping was performed on the Ara h 1, Ara h 2 and Ara h 3allergens by synthesizing each of these proteins in 15 amino acid longoverlapping peptides that were offset from each other by 8 amino acids.The peptides were then probed with a pool of serum IgE from 15 patientswith documented peanut hypersensitivity. This analysis resulted inmultiple IgE binding regions being identified for each allergen. Theexact position of each IgE binding epitope was then determined byre-synthesizing these IgE reactive regions as 10 or 15 amino acid longpeptides that were offset from each other by two amino acids. Thesepeptides were probed with the same pool of serum IgE from peanutsensitive patients as used before. An example of this procedure for eachof the peanut allergens is shown in FIGS. 5-7. FIG. 5 depicts twenty-two10-mer peptides (SEQ ID NOs. 45-66) that span amino acid residues 82-133(SEQ ID NO. 44) of the Ara h 1 allergen (SEQ ID NO. 2). This region ofthe Ara h 1 allergen includes epitopes 4, 5, 6, and 7, as identified inTable 1. FIG. 6 depicts seven 10-mer peptides (SEQ ID NOs. 68-74) thatspan amino acid residues 55-76 (SEQ ID NO. 67) of the Ara h 2 allergen(SEQ ID NO. 4). This region of the Ara h 2 allergen includes epitopes 6and 7 as identified in Table 2. FIG. 7 depicts six 15-mer peptides (SEQID NOs. 76-81) that span amino acid residues 299-321 (SEQ ID NO. 75) ofthe Ara h 3 allergen (SEQ ID NO. 6). This region of the Ara h 3 allergenincludes epitope 4 as identified in Table 3. This analysis revealed thatthere were 23 linear IgE binding epitopes on Ara h 1, 10 epitopes on Arah 2, and 4 epitopes on Ara h 3.

In an effort to determine which, if any, of the epitopes were recognizedby the majority of patients with peanut hypersensitivity, each set ofepitopes identified for the peanut allergens were synthesized and thenprobed individually with serum IgE from 10 different patients. All ofthe patient sera tested recognized multiple epitopes.

Table 1 shows the amino acid sequence and position of each epitopewithin the Ara h 1 protein (SEQ ID NO. 2) of all 23 IgE binding epitopesmapped to this molecule. Table 2 shows the amino acid sequence andposition of each epitope within the Ara h 2 protein (SEQ ID NO. 4) ofall 10 IgE binding epitopes mapped to this molecule. Table 3 shows theamino acid sequence and position of each epitope within the Ara h 3protein (SEQ ID NO. 6) of all 4 IgE binding epitopes mapped to thismolecule.

Four epitopes of the Ara h 1 allergen (peptides 1, 3, 4, 17 of Table 1),three epitopes of the Ara h 2 allergen (peptides 3, 6, 7 of Table 2),and one epitope of the Ara h 3 allergen (peptide 2 of Table 3) wereimmunodominant.

TABLE 1 Ara h 1 IgE binding epitopes EPITOPE AA SEQUENCE POSITIONSEQ ID NO. 1 AKSSPYQKKT 25-34 7 2 QEPDDLKQKA 48-57 8 3 LEYDPRLVYD 65-749 4 GERTRGRQPG 89-98 10 5 PGDYDDDRRQ  97-106 11 6 PRREEGGRWG 107-116 127 REREEDWRQP 123-132 13 8 EDWRRPSHQQ 134-143 14 9 QPRKIRPEGR 143-152 1510 TPGQFEDFFP 294-303 16 11 SYLQEFSRNT 311-320 17 12 FNAEFNEIRR 325-33418 13 EQEERGQRRW 344-353 19 14 DITNPINLRE 393-402 20 15 NNFGKLFEVK409-418 21 16 GTGNLELVAV 461-470 22 17 RRYTARLKEG 498-507 23 18ELHLLGFGIN 525-534 24 19 HRIFLAGDKD 539-548 25 20 IDQIEKQAKD 551-560 2621 KDLAFPGSGE 559-568 27 22 KESHFVSARP 578-587 28 23 PEKESPEKED 597-60629

The underlined portions of each peptide are the smallest IgE bindingsequences as determined by this analysis. All of these sequences can befound in SEQ ID NO. 2.

TABLE 2 Ara h 2 IgE binding epitopes EPITOPE AA SEQUENCE POSITIONSEQ ID NO. 1 HASARQQWEL 15-24 30 2 QWELQGDRRC 21-30 31 3 DRRCQSQLER27-36 32 4 LRPCEQHLMQ 39-48 33 5 KIQRDEDSYE 49-58 34 6 YERDPYSPSQ 57-6635 7 SQDPYSPSPY 65-74 36 8 DRLQGRQQEQ 115-124 37 9 KRELRNLPQQ 127-136 3810 QRCDLDVESG 143-152 39

The underlined portions of each peptide are the smallest IgE bindingsequences as determined by this analysis. All of these sequences can befound in SEQ ID NO. 4.

TABLE 3 Ara h 3 IgE binding epitopes EPITOPE AA SEQUENCE POSITIONSEQ ID NO. 1 IETWNPNNQEFECAG 33-47 40 2 GNIFSGFTPEFLEQA 240-254 41 3VTVRGGLRILSPDRK 279-293 42 4 DEDEYEYDEEDRRRG 303-317 43

The underlined portions of each peptide are the smallest IgE bindingsequences as determined by this analysis. All of these sequences can befound in SEQ ID NO. 6.

Example 6 Modification of Peanut Allergens to Decrease Allergenicity

The major linear IgE binding epitopes of the peanut allergens weremapped using overlapping peptides synthesized on an activated cellulosemembrane and pooled serum IgE from 15 peanut sensitive patients, asdescribed in Example 1. The size of the epitopes ranged from six tofifteen amino acids in length. The amino acids essential to IgE bindingin each of the epitopes were determined by synthesizing duplicatepeptides with single amino acid changes at each position. These peptideswere then probed with pooled serum IgE from 15 patients with peanuthypersensitivity to determine if the changes affected peanut-specificIgE binding. For example, epitope 9 in Table 1 was synthesized with analanine or methionine residue substituted for one of the amino acids andprobed. The following amino acids were substituted (first letter is theone-letter amino acid code for the residue normally at the position, theresidue number, followed by the amino acid that was substituted for thisresidue; the numbers indicate the position of each residue in the Ara h1 protein, SEQ ID NO. 2): Q143A, P144A; R145A; K146A; I147A; R148A;P149A; E150A; G151A; R152A; Q143M; P144M; R145M; K146M; I147M; R148M;P149M; E150M; G151M; R152M. The immunoblot strip containing thewild-type and mutated peptides of epitope 9 showed that binding ofpooled serum IgE to individual peptides was dramatically reduced wheneither alanine or methionine was substituted for each of the amino acidsat positions 144, 145, and 147-150 of Ara h 1 shown in SEQ ID NO. 2.Changes at positions 144, 145, 147, and 148 of Ara h 1 shown in SEQ IDNO. 2 had the most dramatic effect when methionine was substituted forthe wild-type amino acid, resulting in less than 1% of peanut specific1gE binding to these peptides. In contrast, the substitution of analanine for arginine at position 152 of Ara h 1 shown in SEQ ID NO. 2resulted in increased IgE binding. The remaining Ara h 1 epitopes, andthe Ara h 2 and Ara h 3 epitopes, were tested in the same manner and theintensity of IgE binding to each spot was determined as a percentage ofIgE binding to the wild-type peptide. Any amino acid substitution thatresulted in less than 1% of IgE binding when compared to the wild-typepeptide was noted and is indicated in Tables 4-6. Table 4 shows theamino acids that were determined to be critical to IgE binding in eachof the Ara h 1 epitopes. Table 5 shows the amino acids that weredetermined to be critical to IgE binding in each of the Ara h 2epitopes. Table 6 shows the amino acids that were determined to becritical to IgE binding in each of the Ara h 3 epitopes.

This analysis indicated that each epitope could be mutated to a non-IgEbinding-peptide by the substitution of a single amino acid residue.

The results discussed above for Ara h 1, Ara h 2, and Ara h 3demonstrate that once an IgE binding site has been identified, it ispossible to reduce IgE binding to this site by altering a single aminoacid of the epitope. The observation that alteration of a single aminoacid leads to the loss of IgE binding in a population ofpeanut-sensitive individuals is significant because it suggests thatwhile each patient may display a polyclonal IgE reaction to a particularallergen, IgE from different patients that recognize the same epitopemust interact with that epitope in a similar fashion. Besides findingthat many epitopes contained more than one residue critical for IgEbinding, it was also determined that more than one residue type (ala ormet) could be substituted at certain positions in an epitope withsimilar results. This allows for the design of a hypoallergenic proteinthat would be effective at blunting allergic reactions for a populationof peanut sensitive individuals. Furthermore, the creation of a plantproducing a peanut where the IgE binding epitopes of the major allergenshave been removed should prevent the development of peanuthypersensitivity in individuals genetically predisposed to this foodallergy.

TABLE 4 Amino acids critical to IgE binding of Ara h 1 EPITOPEAA SEQUENCE POSITION SEQ ID NO. 1 AKS SPY Q K KT 23-34 7 2 QEP DDL KQKA48-57 8 3 LE YDP RL VY D 65-74 9 4 GE R TR GRQ PG 89-98 10 5 PGDYDD DRRQ  97-106 11 6 PRREE G GRWG 107-116 12 7 REREED W R Q P 123-132 13 8EDW RRP SHQQ 134-143 14 9 Q PR K IR PEGR 143-152 15 10 T P GQ F ED FFP294-303 16 11 S YL Q EF SRNT 311-320 17 12 F NAE F NEIRR 325-334 18 13EQEER G QRRW 344-353 19 14 DIT NP IN L RE 393-402 20 15 NNFGK LF EVK409-418 21 17 RRY TARLKEG 498-507 23 18 EL HL L GFG IN 525-534 24 19HRIFLAGD K D 539-548 25 20 IDQ I EKQ A K D 551-560 26 21 KDLA FPG SGE559-568 27 22 KESHFV S ARP 578-587 28

The Ara h 1 IgE binding epitopes are indicated as the single letteramino acid code. The position of each peptide with respect to the Ara h1 protein (SEQ ID NO. 2) is indicated. The amino acids that, whenaltered, lead to loss of IgE binding are shown as the bold, underlinedresidues. Epitopes 16 and 23 were not included in this study becausethey were recognized by a single patient who was no longer available tothe study. All of these sequences can be found in SEQ ID NO. 2.

TABLE 5 Amino acids critical to IgE binding of Ara h 2 EPITOPEAA SEQUENCE POSITION SEQ ID NO. 1 HASAR Q Q W EL 15-24 30 2 Q W E L Q GDRRC 21-30 31 3 D RR C Q SQL ER 27-36 32 4 L R P CE QH LMQ 39-48 33 5 KIQ RD E D SYE 49-58 34 6 YER DPY SPSQ 57-66 35 7 SQ DPY SPSPY 65-74 36 8DRL QGR QQEQ 115-124 37 9 KR E L RN L PQQ 127-136 38 10 QRC DL D VE SG143-152 39

The Ara h 2 IgE binding epitopes are indicated as the single letteramino acid code. The position of each peptide with respect to the Ara h2 protein (SEQ ID NO. 4) is indicated. The amino acids that, whenaltered, lead to loss of IgE binding are shown as the bold, underlinedresidues. All of these sequences can be found in SEQ ID NO. 4.

TABLE 6 Amino acids critical to IgE binding of Ara h 3 EPITOPEAA SEQUENCE POSITION SEQ ID NO. 1 IETWN PN NQEFECAG 33-47 40 2 GNI F SGF TPE FL EQA 240-254 41 3 VTVRGG L R IL SPDRK 279-293 42 4 DEDEY EYDE EDR RRG 303-317 43

The Ara h 3 IgE binding epitopes are indicated as the single letteramino acid code. The position of each peptide with respect to the Ara h3 protein (SEQ ID NO. 6) is indicated. The amino acids that, whenaltered, lead to loss of IgE binding are shown as the bold, underlined.All of these sequences can be found in SEQ ID NO. 6.

Example 7 A Modified Ara h 2 Protein Binds Less IgE but Similar Amountsof IgG

In order to determine the effect of changes to multiple epitopes withinthe context of the intact allergen, four epitopes (including the threeimmunodominant epitopes) of the Ara h 2 allergen were mutagenized andthe protein produced recombinantly. The amino acids at position 20, 31,60, and 67 of the Ara h 2 protein (shown in SEQ ID NO. 4) were changedto alanine by mutagenizing the gene encoding this protein by standardtechniques. These residues are located in epitopes 1, 3, 6, and 7 andrepresent amino acids critical to IgE binding that were determined inExample 2. The modified and wild-type versions of this protein wereproduced and immunoblot analysis performed using serum from peanutsensitive patients. These results showed that the modified version ofthis allergen bound significantly less IgE than the wild-type version ofthese recombinant proteins but bound similar amounts of IgG.

Example 8 A Modified Ara h 2 Protein Retains the Ability to Stimulate TCells to Proliferate

The modified recombinant Ara h 2 protein described in Example 3 was usedin T cell proliferation assays to determine if it retained the abilityto activate T cells from peanut sensitive individuals. Proliferationassays were performed on T cell lines grown in short-term culturedeveloped from six peanut sensitive patients. T cells lines werestimulated with either 50 μg of crude peanut extract, 10 μg of nativeAra h 2, 10 μg of recombinant wild-type Ara h 2, or 10 μg of modifiedrecombinant Ara h 2 protein and the amount of 3H-thymidine determinedfor each cell line. Results were expressed as the average stimulationindex (SI) which reflected the fold increase in 3H-thymidineincorporation exhibited by cells challenged with allergen when comparedwith media treated controls (FIG. 8).

Example 9 A Modified Ara h 2 Protein Elicits a Smaller Wheal and Flarein Skin Prick Tests of a Peanut Sensitive Individual

The modified recombinant Ara h 2 protein described in Example 3 and thewild-type version of this recombinant protein were used in a skin pricktest of a peanut sensitive individual. Ten micrograms of these proteinswere applied separately to the forearm of a peanut sensitive individual,the skin pricked with a sterile needle, and 10 minutes later any whealand flare that developed was measured. The wheal and flare produced bythe wild-type Ara h 2 protein (8 mm×7 mm) was approximately twice aslarge as that produced by the modified Ara h 2 protein (4 mm×3 mm). Acontrol subject (no peanut hypersensitivity) tested with the sameproteins had no visible wheal and flare but, as expected, gave positiveresults when challenged with histamine. In addition, the test subjectgave no positive results when tested with PBS alone. These resultsindicate that an allergen with only 40% of its IgE binding epitopesmodified (4/10) can give measurable reduction in reactivity in an invivo test of a peanut sensitive patient.

These same techniques can be used with the other known peanut allergens,Ara h 1 (SEQ ID NOs. 1 and 2), Ara h 3 (SEQ ID NOs. 5 and 6), or anyother allergen.

Modifications and variations of the methods and materials describedherein will be obvious to those skilled in the art. Such modificationsand variations are intended to come within the scope of the appendedclaims.

APPENDIX A ACCESSION NO. MW OR ALLERGEN SOURCE SYSTEMATIC AND ORIGINALNAMES KDA SEQ REFERENCES WEED POLLENS Asterales Ambrosia Amb a 1;antigen E 38 C 8, 20 artemisiifolia Amb a 2; antigen K 38 C 8, 21 (shortragweed) Amb a 3; Ra3 11 C 22 Amb a 5; Ra5  5 C 11, 23 Amb a 6; Ra6 10 C24, 25 Amb a 7; Ra7 12 P 26 Amb a ? 11 C 27 Ambrosia trifida Amb t 5;Ra5G   4.4 C 9, 10, 28 (giant ragweed) Artemisia vulgaris Art v 1 27-29C 28A (mugwort) Art v 2 35 P 29 Helianthus annuus Hel a 1 34 — 29a(sunflower) Hel a 2; profilin   15.7 C Y15210 Mercurialis annua Mer a 1;profilin 14-15 C Y13271 GRASS POLLENS Poales Cynodon dactylon Cyn d 1 32C 30, S83343 (Bermuda grass) Cyn d 7 C 31, X91256 Cyn d 12; profilin 14C 31a, Y08390 Dactylis glomerata Dac g 1; AgDg1 32 P 32 (orchard grass)Dac g 2 11 C 33, S45354 Dac g 3 C 33a, U25343 Dac g 5 31 P 34 Holcuslanatus Hol l 1 C Z27084, Z68893 (velvet grass) Lolium perenne Lol p 1;group I 27 C 35, 36 (rye grass) Lol p 2; group II 11 C 37, 37a, X73363Lol p 3; group III 11 C 38 Lol p 5; Lol p IX, 31/35 34, 39 Lol p Ib Lolp 11; trypsin 16 39a inh. Related Phalaris aquatica Pha a 1 C 40, S80654(canary grass) Phleum pratense Phl p 1 27 C X78813 (timothy grass) Phl p2 C 41, X75925 Phl p 4 P 41A Phl p 5; Ag25 32 C 42 Phl p 6 C 43, Z27082Phl p 12; profilin C 44, X77583 Phl p 13; polygalacturonase 55-60 CAJ238848 Poa pratensis Poa p 1; group I 33 P 46 (Kentucky blue Poa p 531/34 C 34, 47 grass) Sorghum halepense Sor h 1 C 48 (Johnson grass)TREE POLLENS Fagales Alnus glutinosa Aln g 1 17 C S50892 (alder) Betulaverrucosa Bet v 1 17 C see list of (birch) isoallergens M65179 Bet v 2;profilin 15 C X79267 Bet v 3 8 C X87153/S54819 Bet v 4 C AF135127 Bet v5; isoflavone reductase   33.5 C homologue Bet v 7; cyclophilin 18 C PP81531 Carpinus betulus Car b 1 17 C 51 (hornbeam) Castanea sativa Cas s1; Bet v 1 homologue 22 P 52 (chestnut) Cas s5; chitinase Corylusavelana Cor a 1 17 C 53 (hazel) Quercus alba Que a 1 17 P 54 (white oak)Cryptomeria japonica Cry j 1 41-45 C 55, 56 (sugi) Cry j 2 C 57, D29772Juniperus ashei Jun a 1 43 P P81294 (mountain cedar) Jun a 3 30 P P81295Juniperus oxycedrus Jun o 2; calmodulin-like 29 C AF031471 (pricklyjuniper) Juniperus sabinoides Jun s 1 50 P 58 (mountain cedar) Juniperusvirginiana Jun v 1 43 P P81825 (eastern red cedar) Oleales Fraxinusexcelsior Fra e 1 20 P 58A (ash) Ligustrum vulgare Lig v 1 20 P 58A(privet) Olea europea Ole e 1; 16 C 59, 60 (olive) Ole e 2; profilin15-18 C 60A Ole e 3;   9.2 60B Ole e 4; 32 P P80741 Ole e 5; superoxidedismutase 16 P P80740 Ole e 6; 10 C U86342 Syringa vulgaris Syr v 1 20 P58A (lilac) MITES Acarus siro Aca s 13; fatty acid-bind.prot.  14* CAJ006774 (mite) Blomia tropicalis Blo t 5; C U59102 (mite) Blo t 12;Bt11a C U27479 Blo t 13; Bt6 fatty acid-binding prot C U58106Dermatophagoides Der p 1; antigen P1 25 C 61 pteronyssinus Der p 2; 14 C62 (mite) Der p 3; trypsin 28/30 C 63 Der p 4; amylase 60 C 64 Der p 5;14 P 65 Der p 6; chymotrypsin 25 C 66 Der p 7; 22-28 C 67 Der p 8;glutathione transferase P 67A Der p 9; collagenolytic serine prot. C 67BDer p 10; tropomyosin 36 Y14906 Der p 14; apolipophorin like p C Eptonp.c. Dermatophagoides Der m 1; 25 P 68 microceras (mite)Dermatophagoides Der f 1; 25 C 69 farinae (mite) Der f 2; 14 C 70, 71Der f 3; 30 C  63 Der f 10; tropomyosin C 72 Der f 11; paramyosin 98 C72a Der f 14; Mag3, apolipophorin C D17686 Euroglyphus maynei Eur m 14;apolipophorin 177  C AF149827 (mite) Lepidoglyphus Lep d 2.0101; 15 C73, 74, 75 destructor Lep d 2.0102; 15 C 75 (storage mite) ANIMALS Bosdomesticus Bos d 2; Ag3, lipocalin 20 C 76, L42867 (domestic cattle) Bosd 4; alpha-lactalbumin   14.2 C M18780 (see also foods) Bos d 5;beta-lactoglobulin   18.3 C X14712 Bos d 6; serum albumin 67 C M73993Bos d 7; immunoglobulin 160  77 Bos d 8; caseins 20-30 77 Canisfamiliaris Can f 1; 25 C 78, 79 (Canis domesticus Can f 2; 27 C 78, 79(dog) Can f ?; albumin C S72946 Equus caballus Equ c 1; lipocalin 25 CU70823 (domestic horse) Equ c 2; lipocali   18.5 P 79A, 79B Felisdomesticus Fel d 1; cat-1 38 C 15 (cat saliva) Mus musculus Mus m 1; MUP19 C 80, 81 (mouse urine) Rattus norvegius Rat n 1 17 C 82, 83 (raturine) FUNGI Ascomycota Dothidiales Alternaria alternata Alt a 1; 28 CU82633 Alt a 2; 25 C U87807, U87808 Alt a 3; heat shock protein 70 CX78222, Alt a 6; ribosomal protein 11 C U87806 Alt a 7; YCP4 protein 22C X78225 Alt a 10; aldehyde dehydrogenase 53 C X78227, P42041 Alt a 11;enolase 45 C U82437 Alt a 12; acid.ribosomal prot P1 11 C X84216Cladosporium Cla h 1; 13 83a, 83b herbarum Cla h 2; 23 83a, 83b Cla h 3;aldehyde dehydrogenase 53 C X78228 Cla h 4; ribosomal protein 11 CX78223 Cla h 5; YCP4 protein 22 C X78224 Cla h 6; enolase 46 C X78226Cla h 12; acid.ribosomal prot P1 11 C X85180 Eurotiales Asp fl 13;alkaline 34 84 serine proteinase Aspergillus Asp f 1; 18 C 83781, S39330Fumigatus Asp f 2; 37 C U56938 Asp f 3; peroxisomal protein 19 C U20722Asp f 4; 30 C AJ001732 Asp f 5; metalloprotease 42 C Z30424 Asp f 6; Mnsuperoxide dismutase   26.5 C U53561 Asp f 7; 12 C AJ223315 Asp f 8;ribosomal protein P2 11 C AJ224333 Asp f 9; 34 C AJ223327 Asp f 10;aspartic protease 34 X85092 Asp f 11; peptidyl-prolyl isom 24 84a Asp f12; heat shock prot. P70 65 C U92465 Asp f 13; alkaline serineproteinase 34 84b Asp f 15; 16 C AJ002026 Asp f 16; 43 C g3643813 Asp f17; 34 C AJ224865 Asp f 18; vacuolar serine 90 84c Asp f ?; 55 P 85 Aspf ?; P 86 Aspergillus niger Asp n 14; beta-xylosidase 105  C AF108944Asp n 18; 34 C 84b vacuolar serine proteinase Asp n ?; 85 C Z84377Aspergillus oryzae Asp o 2; TAKA-amylase A 53 C D00434, M33218 Asp o 13;alkaline serine proteinase 34 C X17561 Penicillium Pen b 13; alkalineserine Proteinase 33 86a brevicompactum Penicillium citrinum Pen c 1;heat shock protein P70 70 C U64207 Pen c 3; peroxisomal membrane 86bprotein Pen c 13; alkaline serine proteinase 33 86a Penicillium notatumPen n 1; N-acetyl 68 87 glucosaminidase Pen n 13; alkaline serineproteinase 34 89 Pen n 18; vacuolar serine proteinase 32 89 Penicilliumoxalicum Pen o 18; vacuolar serine proteinase 34 89 OnygenalesTrichophyton rubrum Tri r 2; C 90 Tri r 4; serine protease C 90Trichophyton Tri t 1; 30 P 91 tonsurans Tri t 4; serine protease 83 C 90Saccharomycetales Candida albicans Cand a 1 40 C 88 Candida boidiniiCand b 2 20 C J04984, J04985 Basidiomycota BasidiolelastomycetesMalassezia furfur Mal f 1; 91a Mal f 2; MF1 peroxisomal 21 C AB011804membrane protein Mal f 3; MF2 peroxisomal 20 C AB011805 membrane proteinMal f 4, 35 C Takesako, p.c. Mal f 5;  18* C AJ011955 Mal f 6;cyclophilin homologue  17* C AJ011956 Basidiomycetes Psilocybe cubensisPsi c 1; 16 91b Psi c 2; cyclophilin Coprinus comatus Cop c 1; 11 CAJ132235 (shaggy cap) Cop c 2; Cop c 3; Brander, p.c. Cop c 5; Brander,p.c. Cop c 7; Brander, p.c. INSECTS Aedes aegyptii Aed a 1; apyrase 68 CL12389 (mosquito) Aed a 2; 37 C M33157 Apis mellifera Api m 1;phospholipase A2 16 C 92 (honey bee) Api m 2; hyaluronidase 44 C 93 Apim 4; melittin  3 C 94 Api m 6; 7-8 P Kettner, p.c. Bombus Bom p 1;phospholipase 16 P 95 pennsylvanicus Bom p 4; protease P 95 (bumble bee)Blattella germanica Bla g 1; Bd90k C 96 (German cockroach) Bla g 2;aspartic protease 36 C Bla g 4; calycin 21 C  97 Bla g 5; glutathionetransf. 22 C 98 Bla g 6; troponin C 27 C  98 Periplaneta americana Per a1; Cr-PII 72-78 C 98A (American cockroach) Per a 3; Cr-PI C Per a 7;tropomyosin 37 C Y14854 Chironomus thummi Chi t 1-9; hemoglobin 16 C 99thummi (midges) Chi t 1.01; component III 16 C P02229 Chi t 1.02;component IV 16 C P02230 Chi t 2.0101; component I 16 C P02221 Chi t2.0102; component IA 16 C P02221 Chi t 3; component II-beta 16 C P02222Chi t 4; component IIIA 16 C P02231 Chi t 5; component VI 16 C P02224Chi t 6.01; component VIIA 16 C P02226 Chi t 6.02; component IX 16 CP02223 Chi t 7; component VIIB 16 C P02225 Chi t 8; component VIII 16 CP02227 Chi t 9; component X 16 C P02228 Dolichovespula Dol m 1;phospholipase A1 35 C 100 maculata Dol m 2; hyaluronidase 44 C 101(white face hornet) Dol m 5; antigen 5 23 C 102, 103 Dolichovespula Dola 5; antigen 5 23 C 104 arenaria (yellow hornet) Polistes annularies Pola 1; phospholipase A1 35 P 105 (wasp) Pol a 2; hyaluronidase 44 P 105Pol a 5; antigen 5 23 C 104 Polistes dominulus Pol d 1; 32-34 C DRHoffman (Mediterranean paper Pol d 4; serine protease DR Hoffman wasp)Pol d 5; P81656 Polistes exclamans Pol e 1; phospholipase A1 34 P 107(wasp) Pol e 5; antigen 5 23 C 104 Polistes fuscatus Pol f 5; antigen 523 C 106 (wasp) Polistes metricus Pol m 5; antigen 5 23 P 106 (wasp)Vespa crabo Vesp c 1; phospholipase 34 P 107 (European hornet) Vesp c5.0101; antigen 5 23 C 106 Vesp c 5.0102; antigen 5 23 C 106 Vespamandarina Vesp m 1.01; DR Hoffman (giant asian hornet) Vesp m 1.02; DRHoffman Vesp m 5; P81657 Vespula flavopilosa Ves f 5; antigen 5 23 C 106(yellowjacket) Vespula germanica Ves g 5; antigen 5 23 C 106(yellowjacket) Vespula maculifrons Ves m 1; phospholipase A1 33.5 C 108(yellowjacket) Ves m 2; hyaluronidase 44 P 109 Ves m 5; antigen 5 23 23104 Vespula Ves p 5; antigen 5 23 C 106 pennsylvanica (yellowjacket)Vespula squamosa Ves s 5; antigen 5 23 C 106 (yellowjacket) Vespulavidua Ves vi 5; 23 C 106 (wasp) Vespula vulgaris Ves v 1; phopholipaseA1 35 C 105A (yellowjacket) Ves v 2; hyaluronidase 44 P 105A Ves v 5;antigen 5 23 C 104 Myrmecia pilosula Myr p 1, C X70256 (Australianjumper Myr p 2; C S81785 ant) Solenopsis geminata Sol g 2; DR Hoffman(tropical fire ant) Sol g 4 DR Hoffman Solenopsis invicta Sol i 2; 13 C110, 111 (fire ant) Sol i 3; 24 C 110 Soli 4; 13 C 110 Solenopsissaevissima Sols 2; DR Hoffman (brazilian fire ant) FOODS Gadus callariasGad c 1; allergen M 12 C 112, 113 (cod) Salmo salar Sals 1; parvalbumin12 C X97824, X97825 (Atlantic salmon) Bos domesticus Bos d 4;alpha-lactalbumin   14.2 C M18780 (domestic cattle) Bos d 5;beta-lactoglobulin   18.3 C X14712 Bos d 6; serum albumin 67 C M73993Bos d 7; immunoglobulin 160  77 Bos d 8; caseins 20-30 77 Gallusdomesticus Gal d 1; ovomucoid 28 C 114, 115 (chicken) Gald 2; ovalbumin44 C 114, 115 Gald 3; conalbumin (Ag22) 78 C 114, 115 Gald 4; lysozyme14 C 114, 115 Gal d 5; serum albumin 69 C X60688 Metapenaeus ensis Met e1; tropomyosin C U08008 (shrimp) Penaeus aztecus Pen a 1; tropomyosin 36P 116 (shrimp) Penaeus indicus Pen i 1; tropomyosin 34 C 117 (shrimp)Todarodes pacificus Tod p 1; tropomyosin 38 P 117A (squid) HaliotisMidae Hal m 1 49 — 117B (abalone) Apium graveolens Api g 1; Bet v 1homologue  16* C Z48967 (celery) Api g 4; profilin AF129423 Api g 5;55/58 P P81943 Brassica juncea Bra j 1; 2S albumin 14 C 118 (orientalmustard) Brassica rapa Bra r 2; prohevein-like protein 25 ? P81729(turnip) Hordeum vulgare Hor v 1; BMAI-1 15 C 119 (barley) Zea mays Zeam 14; lipid transfer prot.  9 P P19656 (maize, corn) Corylus avellanaCor a 1.0401; Bet v 1 17 C AF136945 (hazelnut) homologue Malus domesticaMal d 1; Bet v 1 homologue C X83672 (apple) Mal d 3; lipid transferprotein  9 C Pastorello Pyrus communis Pyr c 1; Bet v 1 homologue 18 CAF05730 (pear) Pyr c 4; profilin 14 C AF129424 Pyr c 5; isoflavonereductase   33.5 C AF071477 homologue Oryza sativa Ory s 1; C U31771(rice) Persea americana Pers a 1; endochitinase 32 C Z78202 (avocado)Prunus armeniaca Pru ar 1; Bet v 1 homologue C U93165 (apricot) Pru ar3; lipid transfer protein  9 P Prunus avium Pru av 1; Bet v 1 homologueC U66076 (sweet cherry) Pru av 2; thaumatin homologue C U32440 Pru av 4;profilin 15 C AF129425 Prunus persica Pru p 3; lipid transfer protein 10P P81402 (peach) Sinapis alba Sin a 1; 2S albumin 14 C 120 (yellowmustard) Glycine max Gly m 1.0101; HPS   7.5 P 121 (soybean) Gly m1.0102; HPS  7 P 121 Gly m 2  8 P A57106 Gly m 3; profilin 14 C AJ223982Arachis hypogaea Ara h 1; vicilin   63.5 C L34402 (peanut) Ara h 2;conglutin 17 C L77197 Ara h 3; glycinin 14 C AF093541 Ara h 4; glycinin37 C AF086821 Ara h 5; profilin 15 C AF059616 Ara h 6; conglutin homolog15 C AF092846 Ara h 7; conglutin homolog 15 C AF091737 Actinidiachinensis Act c 1; cysteine protease 30 P P00785 (kiwi) Solanumtuberosum Sol t 1; patatin 43 P P15476 (potato) Bertholletia excelsa Bere 1; 2S albumin  9 C P04403, M17146 (Brazil nut) Juglans regia Jug r 1;2S albumin 44 C U66866 (English walnut) Jug r 2; vicilin C AF066055Ricinus communis Ric c 1; 2S albumin C P01089 (Castor bean) OTHERSAnisakis simplex Ani s 1 24 P A59069 (nematode) Ani s 2; paramyosin 97 CAF173004 Ascaris suum Asc s 1; 10 P 122 (worm) Aedes aegyptii Aed a 1;apyrase 68 C L12389 (mosquito) Aed a 2; 37 C M33157 Hevea brasiliensisHev b 1; elongation factor 58 P 123, 124 (rubber) Hev b 2;(1,3-glucanase 58 P 123, 124 Hev b 2; (1,3-glucanase 34/36 C 125 Hev b 324 P 126, 127 Hev b 4; component of 100/110/115 P 128 microhelix proteincomplex Hev b 5 16 C U42640 Hev b 6.01 hevein precursor 20 CM36986/p02877 Hev b 6.02 hevein  5 C M36986/p02877 Hev b 6.03 C-terminal14 C M36986/p02877 fragment Hev b 7; patatin homologue 46 C U80598 Hev b8; profilin 14 C Y15042 Hev b 9; enolase 51 C AJ132580/AJ132581 Hev b10; Mn-superoxide 26 C AJ249148 dismut Ctenocephalides felis Cte f 1; —— — felis Cte f 2; M1b 27 C AF231352 (cat flea) Homo sapiens Hom s 1; 73* C Y14314 (human Hom s 2;   10.3* C X80909 autoallergens) Hom s 3;  20.1* C X89985 Hom s 4;  36* C Y17711 Hom s 5;   42.6* C P02538

-   1. Marsh, D. G., and L. R. Freidhoff. 1992. ALBE, an allergen    database. IUIS, Baltimore, Md., Edition 1.0.-   2. Marsh, D. G., L. Goodfriend, T. P. King, H. Lowenstein,    and T. A. E. Platts-Mills. 1986. Allergen nomenclature. Bull WHO    64:767-770.-   3. King, T. P., P. S. Norman, and J. T. Cornell. 1964. Isolation and    characterization of allergen from ragweed pollen. II. Biochemistry    3:458-468.-   4. Lowenstein, H. 1980. Timothy pollen allergens. Allergy    35:188-191.-   5. Aukrust, L. 1980. Purification of allergens in Cladosporium    herbarum. Allergy 35:206-207.-   6. Demerec, M., E. A. Adelberg, A. J. Clark, and P. E.    Hartman. 1966. A proposal for a uniform nomenclature in bacterial    genetics. Genetics 54:61-75.-   7. Bodmer, J. G., E. D. Albert, W. F. Bodmer, B. Dupont, H. A.    Erlich, B. Mach, S. G. E. Marsh, W. R. Mayr, P. Parham, T.    Sasuki, G. M. Th. Schreuder, J. L. Strominger, A. Svejgaard,    and P. I. Terasaki. 1991. Nomenclature for factors of the HLA    system, 1990. Immunogenetics 33:301-309.-   8. Griffith, I. J., J. Pollock, D. G. Klapper, B. L. Rogers,    and A. K. Nault. 1991. Sequence polymorphism of Amb a I and Amb a    II, the major allergens in Ambrosia artemisiifolia (short ragweed).    Int. Arch. Allergy Appl. Immunol. 96:296-304.-   9. Roebber, M., D. G. Klapper, L. Goodfriend, W. B. Bias, S. H. Hsu,    and D. G. Marsh. 1985. Immunochemical and genetic studies of Amb t V    (Ra5G), an Ra5 homologue from giant ragweed pollen. J. Immunol.    134:3062-3069.-   10. Metzler, W. J., K. Valentine, M. Roebber, M. Friedrichs, D. G.    Marsh, and L. Mueller. 1992. Solution structures of ragweed allergen    Amb t V. Biochemistry 31:5117-5127.-   11. Metzler, W. J., K. Valentine, M. Roebber, D. G. Marsh, and L.    Mueller. 1992. Proton resonance assignments and three-dimensional    solution structure of the ragweed allergen Amb a V by nuclear    magnetic resonance spectroscopy. Biochemistry 31:8697-8705.-   12. Goodfriend, L., A. M. Choudhury, J. Del Carpio, and T. P.    King. 1979. Cytochromes C: New ragweed pollen allergens. Fed. Proc.    38:1415.-   13. Ekramoddoullah, A. K. M., F. T. Kisil, and A. H. Sehon. 1982.    Allergenic cross reactivity of cytochrome c from Kentucky bluegrass    and perennial ryegrass pollens. Mol. Immunol. 19:1527-1534.-   14. Ansari, A. A., E. A. Killoran, and D. G. Marsh. 1987. An    investigation of human response to perennial ryegrass (Lolium    perenne) pollen cytochrome c (Lol p X). J. Allergy Clin. Immunol.    80:229-235.-   15. Morgenstern, J. P., I. J. Griffith, A. W. Brauer, B. L.    Rogers, J. F. Bond, M. D. Chapman, and M. Kuo. 1991. Amino acid    sequence of Fel d I, the major allergen of the domestic cat: protein    sequence analysis and cDNA cloning. Proc. Natl. Acad. Sci. USA    88:9690-9694.-   16. Griffith, I. J., S. Craig, J. Pollock, X. Yu, J. P. Morgenstern,    and B. L. Rogers. 1992. Expression and genomic structure of the    genes encoding FdI, the major allergen from the domestic cat. Gene    113:263-268.-   17. Weber, A., L. Marz, and F. Altmann. 1986. Characteristics of the    asparagine-linked oligosaccharide from honey-bee venom phospholipase    A2. Comp. Biochem. Physiol. 83B:321-324.-   18. Weber, A., H. Schroder, K. Thalberg, and L. Marz. 1987. Specific    interaction of IgE antibodies with a carbohydrate epitope of honey    bee venom phospholipase A2. Allergy 42:464-470.-   19. Stanworth, D. R., K. J. Dorrington, T. E. Hugh, K. Reid,    and M. W. Turner. 1990. Nomenclature for synthetic peptides    representative of immunoglobulin chain sequences. Bulletin WHO    68:109-111.-   20. Rafnar, T., I. J. Griffith, M. C. Kuo, J. F. Bond, B. L. Rogers,    and D. G. Klapper. 1991. Cloning of Amb a I (Antigen E), the major    allergen family of short ragweed pollen. J. Biol. Chem. 266:    1229-1236.-   21. Rogers, B. L., J. P. Morgenstern, I. J. Griffith, X. B.    Yu, C. M. Counsell, A. W. Brauer, T. P. King, R. D. Garman,    and M. C. Kuo. 1991. Complete sequence of the allergen Amb a II:    recombinant expression and reactivity with T cells from ragweed    allergic patients. J. Immunol. 147:2547-2552.-   22. Klapper, D. G., L. Goodfriend, and J. D. Capra. 1980. Amino acid    sequence of ragweed allergen Ra3. Biochemistry 19:5729-5734.-   23. Ghosh, B., M. P. Perry, T. Rafnar, and D. G. Marsh. 1993.    Cloning and expression of immunologically active recombinant Amb a V    allergen of short ragweed (Ambrosia artemisiifolia) pollen. J.    Immunol. 150:5391-5399.-   24. Roebber, M., R. Hussain, D. G. Klapper, and D. G. Marsh. 1983.    Isolation and properties of a new short ragweed pollen allergen,    Ra6. J. Immunol. 131:706-711.-   25. Lubahn, B., and D. G. Klapper. 1993. Cloning and    characterization of ragweed allergen Amb a VI (abst). J. Allergy    Clin. Immunol. 91:338.-   26. Roebber, M., and D. G. Marsh. 1991. Isolation and    characterization of allergen Amb a VII from short ragweed pollen. J.    Allergy Clin. Immunol. 87:324.-   27. Rogers, B. L., J. Pollock, D. G. Klapper, and I. J.    Griffith. 1993. Cloning, complete sequence, and recombinant    expression of a novel allergen from short ragweed pollen (abst). J.    Allergy Clin. Immunol. 91:339.-   28. Goodfriend, L., A. M. Choudhury, D. G. Klapper, K. M.    Coulter, G. Dorval, J. DelCarpio, and C. K. Osterland. 1985. Ra5G, a    homologue of Ra5 in giant ragweed pollen: isolation,    HLA-DR-associated activity and amino acid sequence. Mol. Immunol.    22:899-906.-   28A. Breitenbach M, pers. comm.-   29. Nilsen, B. M., K. Sletten, M. O'Neill, B. Smestead Paulsen,    and H. van Halbeek. 1991. Structural analysis of the glycoprotein    allergen Art v II from pollen of mugwort (Artemesia vulgaris). J.    Biol. Chem. 266:2660-2668.-   29A Jimenez A, Moreno C, Martinez J, Martinez A, Bartolome B, Guerra    F, Palacios R 1994. Sensitization to sunflower pollen: only an    occupational allergy? Int Arch Allergy Immunol 105:297-307.-   30. Smith, P. M., Suphioglu, C., Griffith, I. J., Theriault, K.,    Knox, R. B. and Singh, M. B. 1996.-   Cloning and expression in yeast Pichia pastoris of a biologically    active form of Cyn d 1, the major allergen of Bermuda grass    pollen. J. Allergy Clin. Immunol. 98:331-343.-   31. Suphioglu, C., Ferreira, F. and Knox, R. B. 1997. Molecular    cloning and immunological characterisation of Cyn d 7, a novel    calcium-binding allergen from Bermuda grass pollen. FEBS Lett.    402:167-172.-   31a. Asturias J A, Arilla M C, Gomez-Bayon N, Martinez J, Martinez    A, and Palacios R. 1997. Cloning and high level expression of    Cynodon dactylon (Bermuda grass) pollen profilin (Cyn d 12) in    Escherichia coli: purification and characterization of the allergen.    Clin Exp Allergy 27:1307-1313.-   32. Mecheri, S., G. Peltre, and B. David. 1985. Purification and    characterization of a major allergen from Dactylis glomerata pollen:    The Ag Dg 1. Int. Arch. Allergy Appl. Immunol. 78:283-289.-   33. Roberts, A. M., L. J. Bevan, P. S. Flora, I. Jepson, and M. R.    Walker. 1993. Nucleotide sequence of cDNA encoding the Group II    allergen of Cocksfoot/Orchard grass (Dactylis glomerata), Dac g II.    Allergy 48:615-623.-   33a. Guerin-Marchand, C., Senechal, H., Bouin, A. P., Leduc-Brodard,    V., Taudou, G., Weyer, A., Peltre, G. and David, B. 1996. Cloning,    sequencing and immunological characterization of Dac g 3, a major    allergen from Dactylis glomerata pollen. Mol. Immunol. 33:797-806.-   34. Klysner, S., K. Welinder, H. Lowenstein, and F.    Matthiesen. 1992. Group V allergens in grass pollen IV. Similarities    in amino acid compositions and amino terminal sequences of the group    V allergens from Lolium perenne, Poa pratensis and Dactylis    glomerata. Clin. Exp. Allergy 22: 491-497.-   35. Perez, M., G. Y. Ishioka, L. E. Walker, and R. W. Chesnut. 1990.    cDNA cloning and immunological characterization of the rye grass    allergen Lol p I. J. Biol. Chem. 265:16210-16215.-   36. Griffith, I. J., P. M. Smith, J. Pollock, P. Theerakulpisut, A.    Avjioglu, S. Davies, T. Hough, M. B. Singh, R. J. Simpson, L. D.    Ward, and R. B. Knox. 1991. Cloning and sequencing of Lol p I, the    major allergenic protein of rye-grass pollen. FEBS Letters    279:210-215.-   37. Ansari, A. A., P. Shenbagamurthi, and D. G. Marsh. 1989.    Complete amino acid sequence of a Lolium perenne (perennial rye    grass) pollen allergen, Lol p II. J. Biol. Chem. 264:11181-11185.-   37a. Sidoli, A., Tamborini, E., Giuntini, I., Levi, S., Volonte, G.,    Paini, C., De Lalla, C., Siccardi, A. G., Baralle, F. E.,    Galliani, S. and Arosio, P. 1993. Cloning, expression, and    immunological characterization of recombinant Lolium perenne    allergen Lol p II. J. Biol. Chem. 268:21819-21825.-   38. Ansari, A. A., P. Shenbagamurthi, and D. G. Marsh. 1989.    Complete primary structure of a Lolium perenne (perennial rye grass)    pollen allergen, Lol p III: Comparison with known Lol p I and II    sequences. Biochemistry 28:8665-8670.-   39. Singh, M. B., T. Hough, P. Theerakulpisut, A. Avjioglu, S.    Davies, P. M. Smith, P. Taylor, R. J. Simpson, L. D. Ward, J.    McCluskey, R. Puy, and R. B. Knox. 1991. Isolation of cDNA encoding    a newly identified major allergenic protein of rye-grass pollen:    Intracellular targeting to the amyloplost. Proc. Natl. Acad. Sci.    88:1384-1388.-   39a. van Ree R, Hoffman D R, van Dijk W, Brodard V, Mahieu K,    Koeleman C A, Grande M, van Leeuwen W A, Aalberse R C. 1995. Lol p    XI, a new major grass pollen allergen, is a member of a family of    soybean trypsin inhibitor-related proteins. J Allergy Clin Immunol    95:970-978.-   40. Suphioglu, C. and Singh, M. B. 1995. Cloning, sequencing and    expression in Escherichia coli of Pha a 1 and four isoforms of Pha a    5, the major allergens of canary grass pollen. Clin. Exp. Allergy    25:853-865.-   41. Dolecek, C., Vrtala, S., Laffer, S., Steinberger, P., Kraft, D.,    Scheiner, O. and Valenta, R. 1993. Molecular characterization of Ph1    p II, a major timothy grass (Phleum pratense) pollen allergen. FEBS    Lett. 335:299-304.-   41A. Fischer S, Grote M, Fahlbusch B, Muller W D, Kraft D,    Valenta R. 1996. Characterization of Ph1 p 4, a major timothy grass    (Phleum pratense) pollen allergen. J Allergy Clin Immunol    98:189-198.-   42. Matthiesen, F., and H. Lowenstein. 1991. Group V allergens in    grass pollens. I. Purification and characterization of the group V    allergen from Phleum pratense pollen, Ph1 p V. Clin. Exp. Allergy    21:297-307.-   43. Petersen, A., Bufe, A., Schramm, G., Schlaak, M. and    Becker, W. M. 1995. Characterization of the allergen group VI in    timothy grass pollen (Ph1 p 6). II. cDNA cloning of Ph1 p 6 and    structural comparison to grass group V. Int. Arch. Allergy Immunol.    108:55-59.-   44. Valenta, R., Ball, T., Vrtala, S., Duchene, M., Kraft, D. and    Scheiner, O. 1994. cDNA cloning and expression of timothy grass    (Phleum pratense) pollen profilin in Escherichia coli: comparison    with birch pollen profilin. Biochem. Biophys. Res. Commun.    199:106-118.-   46. Esch, R. E., and D. G. Klapper. 1989. Isolation and    characterization of a major cross-reactive grass group I allergenic    determinant. Mol. Immunol. 26:557-561.-   47. Olsen, E., L. Zhang, R. D. Hill, F. T. Kisil, A. H. Sehon,    and S. Mohapatra. 1991. Identification and characterization of the    Poa p IX group of basic allergens of Kentucky bluegrass pollen. J.    Immunol. 147:205-211.-   48. Avjioglu, A., M. Singh, and R. B. Knox. 1993. Sequence analysis    of Sor h I, the group I allergen of Johnson grass pollen and it    comparison to rye-grass Lol p I (abst). J. Allergy Clin. Immunol.    91:340.-   51. Larsen, J. N., P. Str^(o)man, and H. Ipsen. 1992. PCR based    cloning and sequencing of isogenes encoding the tree pollen major    allergen Car b I from Carpinus betulus, hornbeam. Mol. Immunol.    29:703-711.-   52. Kos T, Hoffmann-Sommergruber K, Ferreira F, Hirschwehr R, Ahorn    H, Horak F, Jager S, Sperr W, Kraft D, Scheiner O. 1993.    Purification, characterization and N-terminal amino acid sequence of    a new major allergen from European chestnut pollen—Cas s 1. Biochem    Biophys Res Commun 196:1086-92.-   53. Breiteneder, H., F. Ferreira, K. Hoffman-Sommergruber, C.    Ebner, M. Breitenbach, H. Rumpold, D. Kraft, and O. Scheiner. 1993.    Four recombinant isoforms of Cor a I, the major allergen of hazel    pollen. Europ. J. Biochem. 212:355-362.-   54. Ipsen, H., and B. C. Hansen. 1991. The NH2-terminal amino acid    sequence of the immunochemically partial identical major allergens    of alder (Alnus glutinosa) Aln g I, birch (Betula verrucosa) Bet v    I, hornbeam (Carpinus betulus) Car b I and oak (Quercus alba) Que a    I pollens. Mol. Immunol. 28:1279-1288.-   55. Taniai, M., S. Ando, M. Usui, M. Kurimoto, M. Sakaguchi, S.    Inouye, and T. Matuhasi. 1988. N-terminal amino acid sequence of a    major allergen of Japanese cedar pollen (Cry j I). FEBS Lett.    239:329-332.-   56. Griffith, I. J., A. Lussier, R. Garman, R. Koury, H. Yeung,    and J. Pollock. 1993. The cDNA cloning of Cry j I, the major    allergen of Cryptomeria japonica (Japanese cedar) (abst). J. Allergy    Clin. Immunol. 91:339.-   57. Sakaguchi, M., S. Inouye, M. Taniai, S. Ando, M. Usui, and T.    Matuhasi. 1990. Identification of the second major allergen of    Japanese cedar pollen. Allergy 45:309-312.-   58 Gross G N, Zimburean J M, Capra J D 1978. Isolation and partial    characterization of the allergen in mountain cedar pollen. Scand J    Immunol 8:437-41-   58A Obispo T M, Melero J A, Carpizo J A, Carreira J, Lombardero    M 1993. The main allergen of Olea europaea (Ole e I) is also present    in other species of the oleaceae family. Clin Exp Allergy    23:311-316.-   59. Cardaba, B., D. Hernandez, E. Martin, B. de Andres, V. del    Pozo, S. Gallardo, J. C. Fernandez, R. Rodriguez, M. Villalba, P.    Palomino, A. Basomba, and C. Lahoz. 1993. Antibody response to olive    pollen antigens: association between HLA class II genes and IgE    response to Ole e I (abst). J. Allergy Clin. Immunol. 91:338.-   60. Villalba, M., E. Batanero, C. Lopez-Otin, L. M. Sanchez, R. I.    Monsalve, M. A. Gonzalez de la Pena, C. Lahoz, and R.    Rodriguez. 1993. Amino acid sequence of Ole e I, the major allergen    from olive tree pollen (Olea europaea). Europ. J. Biochem.    216:863-869.-   60A. Asturias J A, Arilla M C, Gomez-Bayon N, Martinez J, Martinez    A, Palacios R 1997. Cloning and expression of the panallergen    profilin and the major allergen (Ole e 1) from olive tree pollen. J    Allergy Clin Immunol 100:365-372.-   60B. Batanero E, Villalba M, Ledesma A Puente X S,    Rodriguez R. 1996. Ole e 3, an olive-tree allergen, belongs to a    widespread family of pollen proteins. Eur J Biochem 241: 772-778.-   61. Chua, K. Y., G. A. Stewart, and W. R. Thomas. 1988. Sequence    analysis of cDNA encoding for a major house dust mite allergen, Der    p I. J. Exp. Med. 167:175-182.-   62. Chua, K. Y., C. R. Doyle, R. J. Simpson, K. J. Turner, G. A.    Stewart, and W. R. Thomas. 1990. Isolation of cDNA coding for the    major mite allergen Der p II by IgE plaque immunoassay. Int. Arch.    Allergy Appl. Immunol. 91:118-123.-   63. Smith W A, Thomas W R. 1996. Comparative analysis of the genes    encoding group 3 allergens from Dermatophagoides pteronyssinus and    Dermatophagoides farinae. Int Arch Allergy Immunol 109: 133-40.-   64. Lake, F. R., L. D. Ward, R. J. Simpson, P. J. Thompson,    and G. A. Stewart. 1991. House dust mite-derived amylase:    Allergenicity and physicochemical characterisation. J. Allergy Clin.    Immunol. 87:1035-1042.-   65. Tovey, E. R., M. C. Johnson, A. L. Roche, G. S. Cobon, and B. A.    Baldo. 1989. Cloning and sequencing of a cDNA expressing a    recombinant house dust mite protein that binds human IgE and    corresponds to an important low molecular weight allergen. J. Exp.    Med. 170:1457-1462.-   66. Yasueda, H., T. Shida, T. Ando, S. Sugiyama, and H.    Yamakawa. 1991. Allergenic and proteolytic properties of fourth    allergens from Dermatophagoides mites. In: “Dust Mite Allergens and    Asthma. Report of the 2nd international workshop” A. Todt, Ed., UCB    Institute of Allergy, Brussels, Belgium, pp. 63-64.-   67. Shen, H.-D., K.-Y. Chua, K.-L. Lin, K.-H. Hsieh, and W. R.    Thomas. 1993. Molecular cloning of a house dust mite allergen with    common antibody binding specificities with multiple components in    mite extracts. Clin. Exp. Allergy 23:934-40.-   67A. O'Neil G M, Donovan G R, Baldo B A. 1994. Cloning and    characterisation of a major allergen of the house dust mite    Dermatophagoides pteronyssinus, homologous with glutathione    S-transferase. Biochim Biophys Acta, 1219:521-528.-   67B. King C, Simpson R J, Moritz R L, Reed G E, Thompson P J,    Stewart G A. 1996. The isolation and characterization of a novel    collagenolytic serine protease allergen (Der p 9) from the dust mite    Dermatophagoides pteronyssinus. J Allergy Clin Immunol 98:739-47.-   68. Lind P, Hansen O C, Horn N. 1988. The binding of mouse hybridoma    and human IgE antibodies to the major fecal allergen, Der p I of D.    pteronyssinus. J. Immunol. 140:4256-4262.-   69. Dilworth, R. J., K. Y. Chua, and W. R. Thomas. 1991. Sequence    analysis of cDNA coding for a mojor house dust allergen Der f I.    Clin. Exp. Allergy 21:25-32.-   70. Nishiyama, C., T. Yunki, T. Takai, Y. Okumura, and H.    Okudaira. 1993. Determination of three disulfide bonds in a major    house dust mite allergen, Der f II. Int. Arch. Allergy Immunol.    101:159-166.-   71. Trudinger, M., K. Y. Chua, and W. R. Thomas. 1991. cDNA encoding    the major dust mite allergen Der f II. Clin. Exp. Allergy 21:33-38.-   72. Aki T, Kodama T, Fujikawa A, Miura K, Shigeta S, Wada T, Jyo T,    Murooka Y, Oka S, Ono K. 1995. Immunochemical characterisation of    recombinant and native tropomyosins as a new allergen from the house    dust mite Dermatophagoides farinae. J Allergy Clin Immunol 96:74-83.-   73. van Hage-Hamsten, M., T. Bergman, E Johansson, B. Persson, H.    Jornvall, B. Harfast, and S. G. O. Johansson. 1993. N-terminal amino    acid sequence of major allergen of the mite lepidoglyphus destructor    (abst). J. Allergy Clin. Immunol. 91:353.-   74. Varela J, Ventas P, Carreira J, Barbas J A, Gimenez-Gallego G,    Polo F. Primary structure of Lep d I, the main Lepidoglyphus    destructor allergen. Eur J Biochem 225:93-98, 1994.-   75. Schmidt M, van der Ploeg I, Olsson S, van Hage Hamsten M. The    complete cDNA encoding the Lepidoglyphus destructor major allergen    Lep d 1. FEBS Lett 370:11-14, 1995.-   76. Rautiainen J, Rytkonen M, Pelkonen J, Pentikainen J, Perola O,    Virtanen T, Zeiler T, Mantyjarvi R. BDA20, a major bovine dander    allergen characterized at the sequence level is Bos d 2. Submitted.-   77. Gjesing B, Lowenstein H. Immunochemistry of food antigens. Ann    Allergy 53:602, 1984.-   78. de Groot, H., K. G. H. Goei, P. van Swieten, and R. C. Aalberse.    1991 Affinity purification of a major and a minor allergen from dog    extract: Serologic activity of affinity-purified Can f I and Can f    I-depleted extract. J. Allergy Clin. Immunol. 87:1056-1065.-   79. Konieczny, A. Personal communication; Immunologic Pharmaceutical    Corp.-   79A. Bulone, V. 1998. Separation of horse dander allergen proteins    by two-dimensional electrophoresis. Molecular characterisation and    identification of Equ c 2.0101 and Equ c 2.0102 as lipocalin    proteins. Eur J Biochem 253:202-211.-   79B. Swiss-Prot acc. P81216, P81217.-   80. McDonald, B., M. C. Kuo, J. L. Ohman, and L. J.    Rosenwasser. 1988. A 29 amino acid peptide derived from rat alpha 2    euglobulin triggers murine allergen specific human T cells    (abst). J. Allergy Clin. Immunol. 83:251.-   81. Clarke, A. J., P. M. Cissold, R. A. Shawi, P. Beattie, and J.    Bishop. 1984. Structure of mouse urinary protein genes: differential    splicing configurations in the 3′-non-coding region. EMBO J    3:1045-1052.-   82. Longbottom, J. L. 1983. Characterization of allergens from the    urines of experimental animals. McMillan Press, London, pp. 525-529.-   83. Laperche, Y., K. R. Lynch, K. P. Dolans, and P. Feigelsen. 1983.    Tissue-specific control of alpha 2u globulin gene expression:    constitutive synthesis in submaxillary gland. Cell 32:453-460.-   83A. Aukrust L, Borch S M. 1979. Partial purification and    characterization of two Cladosporium herbarum allergens. Int Arch    Allergy Appl Immunol 60:68-79.-   83B. Sward-Nordmo M, Paulsen B S, Wold J K. 1988. The glycoprotein    allergen Ag-54 (Cla h II) from Cladosporium herbarum. Structural    studies of the carbohydrate moiety. Int Arch Allergy Appl Immunol    85:288-294.-   84. Shen, et al. J. Allergy Clin. Immunol. 103:S157, 1999.-   84A. Crameri R. Epidemiology and molecular basis of the involvement    of Aspergillus fumigatus in allergic diseases. Contrib. Microbiol.    Vol. 2, Karger, Basel (in press).-   84B. Shen, et al. (manuscript submitted), 1999-   84C. Shen H D, Ling W L, Tan M F, Wang S R, Chou H, Han S I H.    Vacuolar serine proteinase: A major allergen of Aspergillus    fumigatus. 10th International Congress of Immunology, Abstract,    1998.-   85. Kumar, A., L. V. Reddy, A. Sochanik, and V. P. Kurup. 1993.    Isolation and characterization of a recombinant heat shock protein    of Aspergillus fumigatus. J. Allergy Clin. Immunol. 91:1024-1030.-   86. Teshima, R., H. Ikebuchi, J. Sawada, S. Miyachi, S. Kitani, M.    Iwama, M. Irie, M. Ichinoe, and T. Terao. 1993. Isolation and    characterization of a major allergenic component (gp55) of    Aspergillus fumigatus. J. Allergy Clin. Immunol. 92:698-706.-   86A. Shen H D, Lin W L, Tsai J J, Liaw S F, Han S H. 1996.    Allergenic components in three different species of Penicillium:    crossreactivity among major allergens. Clin Exp Allergy 26:444-451.-   86B. Shen, et al. Abstract; The XVIII Congress of the European    Academy of Allergology and Clinical Immunology, Brussels, Belgium,    3-7 Jul. 1999.-   87. Shen H D, Liaw S F, Lin W L, Ro L H, Yang H L, Han S H. 1995.    Molecular cloning of cDNA coding for the 68 kDa allergen of    Penicillium notatum using MoAbs. Clin Exp Allergy 25:350-356.-   88. Shen, H. D., K. B. Choo, H. H. Lee, J. C. Hsieh, and S. H.    Han. 1991. The 40 kd allergen of Candida albicans is an alcohol    dehydrogenease: molecular cloning and immunological analysis using    monoclonal antibodies. Clin. Exp. Allergy 21:675-681.-   89. Shen, et al. Clin. Exp. Allergy (in press), 1999.-   90. Woodfolk J A, Wheatley L M, Piyasena R V, Benjamin D C,    Platts-Mills T A. 1998. Trichophyton antigens associated with IgE    antibodies and delayed type hypersensitivity. Sequence homology to    two families of serine proteinases. J Biol Chem 273:29489-96.-   91. Deuell, B., L. K. Arruda, M. L. Hayden, M. D. Chapman    and T. A. E. Platts-Mills. 1991. Trichophyton tonsurans    Allergen I. J. Immunol. 147:96-101.-   91A. Schmidt M, Zargari A, Holt P, Lindbom L, Hellman U, Whitley P,    van der Ploeg I, Harfast B, Scheynius A. 1997. The complete cDNA    sequence and expression of the first major allergenic protein of    Malassezia furfur, Mal f 1. Eur J Biochem 246:181-185.-   91B. Homer W E, Reese G, Lehrer S B. 1995. Identification of the    allergen Psi c 2 from the basidiomycete Psilocybe cubensis as a    fungal cyclophilin. Int Arch Allergy Immunol 107:298-300.-   92. Kuchler, K., M. Gmachl, M. J. Sippl, and G. Kreil. 1989.    Analysis of the cDNA for phospholipase A2 from honey bee venom    glands: The deduced amino acid sequence reveals homology to the    corresponding vertebrate enzymes. Eur. J. Biochem. 184:249-254.-   93. Gmachl, M., and G. Kreil. 1993. Bee venom hyaluronidase is    homologous to a membrane protein of mammalian sperm. Proc. Natl.    Acad. Sci. USA 90:3569-3573.-   94. Habermann, E. 1972. Bee and wasp venoms. Science 177:314-322.-   95. Jacobson, R. S., and D. R. Hoffman. 1993. Characterization of    bumblebee venom allergens (abst). J. Allergy Clin. Immunol. 91:187.-   96. Arruda L K, Vailes L D, Mann B J, Shannon J, Fox J W, Vedvick T    S, Hayden M L, Chapman M D. Molecular cloning of a major cockroach    (Blattella germanica) allergen, Bla g 2. Sequence homology to the    aspartic proteases. J Biol Chem 270:19563-19568, 1995.-   97. Arruda L K, Vailes L D, Hayden M L, Benjamin D C, Chapman M D.    Cloning of cockroach allergen, Bla g 4, identifies ligand binding    proteins (or calycins) as a cause of IgE antibody responses. J Biol    Chem 270:31196-31201, 1995.-   98. Arruda L K, Vailes L D, Benjamin D C, Chapman M D. Molecular    cloning of German Cockroach (Blattella germanica) allergens. Int    Arch Allergy Immunol 107:295-297, 1995.-   98A. Wu C H, Lee M F, Liao S C. 1995. Isolation and preliminary    characterization of cDNA encoding American cockroach allergens. J    Allergy Clin Immunol 96: 352-9.-   99. Mazur, G., X. Baur, and V. Liebers. 1990. Hypersensitivity to    hemoglobins of the Diptera family Chironomidae: Structural and    functional studies of their immunogenic/allergenic sites. Monog.    Allergy 28:121-137.-   100. Soldatova, L., L. Kochoumian, and T. P. King. 1993. Sequence    similarity of a hornet (D. maculata) venom allergen phospholipase A1    with mammalian lipases. FEBS Letters 320:145-149.-   101. Lu, G., L. Kochoumian and T. P. King. Whiteface hornet venom    allergen hyaluronidase: cloning and its sequence similarity with    other proteins (abst.). 1994. J. Allergy Clin. Immunol. 93:224.-   102. Fang, K. S. F., M. Vitale, P. Fehlner, and T. P. King. 1988.    cDNA cloning and primary structure of a white-faced hornet venom    allergen, antigen 5. Proc. Natl. Acad. Sci., USA 85:895-899.-   103. King, T. P., D. C. Moran, D. F. Wang, L. Kochoumian, and B. T.    Chait. 1990. Structural studies of a hornet venom allergen antigen    5, Dol m V and its sequence similarity with other proteins. Prot.    Seq. Data Anal. 3:263-266.-   104. Lu, G., M. Villalba, M. R. Coscia, D. R. Hoffman, and T. P.    King. 1993. Sequence analysis and antigen cross reactivity of a    venom allergen antigen 5 from hornets, wasps and yellowjackets. J.    Immunol. 150: 2823-2830.-   105. King, T. P. and Lu, G. 1997. Unpublished data.-   105A. King T P, Lu G, Gonzalez M, Qian N and Soldatova L. 1996.    Yellow jacket venom allergens, hyaluronidase and phospholipase:    sequence similarity and antigenic cross-reactivity with their hornet    and wasp homologs and possible implications for clinical allergy. J.    Allergy Clin. Immunol. 98:588-600.-   106. Hoffman, D. R. 1993. Allergens in hymenoptera venom XXV: The    amino acid sequences of antigen 5 molecules and the structural basis    of antigenic cross-reactivity. J. Allergy Clin. Immunol. 92:707-716.-   107. Hoffman, D. R. 1992. Unpublished data.-   108. Hoffman, D. R. 1993. The complete amino acid sequence of a    yellowjacket venom phospholipase (abst). J. Allergy Clin. Immunol.    91:187.-   109. Jacobson, R. S., D. R. Hoffman, and D. M. Kemeny. 1992. The    cross-reactivity between bee and vespid hyaluronidases has a    structural basis (abst). J. Allergy Clin. Immunol. 89:292.-   110. Hoffman, D. R. 1993. Allergens in Hymenoptera venom XXIV: The    amino acid sequences of imported fire ant venom allergens Sol i II,    Sol i III, and Sol i IV. J. Allergy Clin. Immunol. 91:71-78.-   111. Schmidt, M., R. B. Walker, D. R. Hoffman, and T. J.    McConnell. 1993. Nucleotide sequence of cDNA encoding the fire ant    venom protein Sol i II. FEBS Letters 319:138-140.-   112. Elsayed S, Bennich H. The primary structure of Allergen M from    cod. Scand J Immunol 3:683-686, 1974.-   113. Elsayed S, Aas K, Sletten K, Johansson S G O. Tryptic cleavage    of a homogeneous cod fish allergen and isolation of two active    polypeptide fragments. Immunochemistry 9:647-661, 1972.-   114. Hoffman, D. R. 1983. Immunochemical identification of the    allergens in egg white. J. Allergy Clin. Immunol. 71:481-486.-   115. Langeland, T. 1983. A clinical and immunological study of    allergy to hen's egg white. IV. specific IgE antibodies to    individual allergens in hen's egg white related to clinical and    immunological parameters in egg-allergic patients. Allergy    38:493-500.-   116. Daul, C. B., M. Slattery, J. E. Morgan, and S. B. Lehrer. 1993.    Common crustacea allergens: identification of B cell epitopes with    the shrimp specific monoclonal antibodies. In: “Molecular Biology    and Immunology of Allergens” (D. Kraft and A. Sehon, eds.). CRC    Press, Boca Raton. pp. 291-293.-   117. K. N. Shanti, B. M. Martin, S. Nagpal, D. D. Metcalfe, P. V.    Subba Rao. 1993. Identification of tropomyosin as the major shrimp    allergen and characterization of its IgE-binding epitopes. J.    Immunol. 151:5354-5363.-   117A. M. Miyazawa, H. Fukamachi, Y. Inagaki, G. Reese, C. B.    Daul, S. B. Lehrer, S. Inouye, M. Sakaguchi. 1996. Identification of    the first major allergen of a squid (Todarodes pacificus). J.    Allergy Clin. Immunol. 98:948-953.-   117B A. Lopata et al. 1997. Characteristics of hypersensitivity    reactions and identification of a uniques 49 kDa IgE binding protein    (Hal-m-1) in Abalone (Haliotis midae). J. Allergy Clin. Immunol.    Submitted-   118. Monsalve, R. I., M. A. Gonzalez de la Pena, L.    Menendez-Arias, C. Lopez-Otin, M. Villalba, and R. Rodriguez. 1993.    Characterization of a new mustard allergen, Bra j IE. Detection of    an allergenic epitope. Biochem. J. 293:625-632.-   119. Mena, M., R. Sanchez-Monge, L. Gomez, G. Salcedo, and P.    Carbonero. 1992. A major barley allergen associated with baker's    asthma disease is a glycosylated monomeric inhibitor of insect    alpha-amylase: cDNA cloning and chromosomal location of the gene.    Plant Molec. Biol. 20:451-458.-   120. Menendez-Arias, L., I. Moneo, J. Dominguez, and R.    Rodriguez. 1988. Primary structure of the major allergen of yellow    mustard (Sinapis alba L.) seed, Sin a I. Eur. J. Biochem.    177:159-166.-   121. Gonzalez R, Varela J, Carreira J, Polo F. Soybean hydrophobic    protein and soybean hull allergy. Lancet 346:48-49, 1995.-   122. Christie, J. F., B. Dunbar, I. Davidson, and M. W.    Kennedy. 1990. N-terminal amino acid sequence identity between a    major allergen of Ascaris lumbricoides and Ascaris suum and    MHC-restricted IgE responses to it. Immunology 69:596-602.-   123. Czuppon A B, Chen Z, Rennert S, Engelke T, Meyer H E, Heber M,    Baur X. The rubber elongation factor of rubber trees (Hevea    brasiliensis) is the major allergen in latex. J Allergy Clin Immunol    92:690-697, 1993.-   124. Attanayaka D P S T G, Kekwick R G O, Franklin F C H. 1991.    Molecular cloning and nucleotide sequencing of the rubber elongation    factor gene from hevea brasiliensis. Plant Mol Biol 16:1079-1081.-   125. Chye M L, Cheung K Y. 1995. (1,3-glucanase is highly expressed    in Laticifers of Hevea brasiliensis. Plant Mol Biol 26:397-402.-   126. Alenius H, Palosuo T, Kelly K, Kurup V, Reunala T,    Makinen-Kiljunen S, Turjanmaa K Fink J. 1993. IgE reactivity to    14-kD and 27-kD natural rubber proteins in Latex-allergic children    with Spina bifida and other congenital anomalies. Int Arch Allergy    Immunol 102:61-66.-   127. Yeang H Y, Cheong K F, Sunderasan E, Hamzah S, Chew N P, Hamid    S, Hamilton R G, Cardosa M J. 1996. The 14.6 kD (REF, Hey b 1) and    24 kD (Hey b 3) rubber particle proteins are recognized by IgE from    Spina Bifida patients with Latex allergy. J Allerg Clin Immunol in    press.-   128. Sunderasan E, Hamzah S, Hamid S, Ward M A, Yeang H Y, Cardosa    M J. 1995. Latex B-serum (-1,3-glucanase (Hey b 2) and a component    of the microhelix (Hey b 4) are major Latex allergens. J nat Rubb    Res 10:82-99.

We claim: 1-33. (canceled)
 34. A composition comprising: (a) dead E.coli cells that have expressed and encapsulated therein a non-secreted,recombinant version of an allergen protein; and (b) a pharmaceuticallyacceptable carrier appropriate for rectal, vaginal, nasal, oral, buccal,or mucosal delivery, the composition being formulated for rectal,vaginal, nasal, oral, buccal, or mucosal delivery.